key: cord-267540-9p4rky4c authors: joseph, iype title: middle east respiratory syndrome corona virus (mers cov): the next steps date: 2015-03-26 journal: j public health policy doi: 10.1057/jphp.2015.9 sha: doc_id: 267540 cord_uid: 9p4rky4c developing countries are at risk of importing middle east respiratory syndrome corona virus (mers cov) from the middle east. hospitals in the middle east currently reporting the disease are staffed by immigrants. in the current hot spots for mers cov a sizeable portion of the population is from other countries, but many of these countries have yet to detect any importation of mers cov. to assess the disease transmission in these countries, supplemental surveillance strategies are urgently needed beyond the currently recommended measures. a few strategies to address the situation are: (i) improving preparedness with enhanced surveillance in particular regions; (ii) targeting certain sentinel groups for surveillance in hot spots; and (iii) limited use of serosurveillance. recovered, immune patients can be employed to give patient care during outbreaks. saudi arabia reported the first case of middle east respiratory syndrome corona virus (mers-cov) in september, 2012. 1 also caused by a corona virus, it is not unlike sars. world over, a total of 965 laboratory-confirmed cases of infection, including at least 357 related deaths have been reported to the world health organization (who) as of 3 february 2015. 2 in addition to outbreaks in the arabian peninsula, sporadic cases have been imported to europe, africa, asia, and north america in returning travellers. no sustained transmission has been reported outside the arabian peninsula. evidence point to a reservoir for the virus among both bats 3 and camels, 4 but the issue is not yet settled. 5 who coordinates international surveillance. deaths, sick persons who recover, and asymptomatic carriers continue to be found. cases arise from unknown sources in the community and from within hospitals in the middle east region that have cared for laboratory-proven cases. surveillance systems in parts of the world outside the arabian peninsula remain deliberately inactive. how will the situation evolve? will the disease be extinguished at its source? will the current state of geographically limited transmission persist indefinitely? will it spread far and wide, immediately or after a delay? 6 exportation is being closely watched, but might the virus find another niche environment where it can successfully establish sustained transmission? clinically, 7 mers cov presents with symptoms of lower respiratory tract disease (fever, cough, dyspnoea, and chest pain), sore throat, myalgia, malaise, and gastro-intestinal symptoms, such as diarrhoea, vomiting and abdominal pain. complications described in fatal cases are hyperkalaemia with associated ventricular tachycardia, disseminated intravascular coagulation leading to cardiac arrest, pericarditis and multi-organ failure. a large proportion of the severely ill patients require mechanical ventilation. fatalities are more in those with co-morbid conditions and an age over 60 years. preparedness to face the mers cov threat in tropical developing countries is limited. experience of managing pandemic h1n1 (gathered from the prior threat of sars) might help prepare countries. [8] [9] [10] despite persisting knowledge gaps about transmission dynamics rational preparedness can begin. preparedness can begin in priority countries and in geographical locations with increased potential for importation. mers outbreaks linked to hospitals and to health care centre visits have been documented many times in the middle east. hospitals in the middle-east employ immigrant health staff, especially from india 11 and the philippines. 11, 12 in addition, much of the population includes labour immigrants 13 from pakistan, sri lanka, egypt, bangladesh, and indonesia. a look at where immigrants come from and their travel back to their native lands in the past 28 months would demarcate the geographical areas with potential for importation. a useful technique has already used hajj pilgrim data 14 (from the population of muslims who come from all over the world to mecca each year). the data on hospital employees can be extracted from the immigration departments of the affected middle-east countries. who might collate this important kind of data. health-care workers 15 are a high risk group to acquire mers cov. ten studies that addressed this issue have found that, among those infected, 24.4 per cent (90/369) were health-care workers. 16 the role of the asymptomatic health-care workers in the incubation period and of minimally sick health-care workers who continue to offer clinical services thus propagating the disease transmission have also been documented. 16 in addition, the hajj pilgrims have spread the disease. among the travellers, currently symptomatic individuals who satisfy the who case definition are being targeted for virological surveillance. travelling health-care workers, irrespective of whether they are symptomatic can be selected for virological testing. what is happening with mers cov transmission in countries with high likelihood of importation? this has not yet been adequately assessed. using the current strategy of virological testing of patients fulfilling the who case definition, india, pakistan, egypt, bangladesh, philippines, sri lanka, and indonesia 13 (countries having sizable numbers of health workers employed in the middle-east) have not yet detected more than one case each. is this because of the absence of importation or to surveillance systems in these countries? perhaps an alternate strategy is needed to detect arrival of the virus into these countries. as virological testing is not readily available in less developed countries they might use serological methods. even with the limits of serological tests, results from multiple tests may yield important information. 17 indigenously acquired sero-positivity against mers cov would currently be a good indicator of importation. overcrowded hospitals 18 have been shown to provoke nosocomial outbreaks of airborne diseases, like sars. particular characteristics enhance transmission, possibly through aerosol and contact: distance between beds of ⩽1m; staff continuing work while symptomatic; and host patient requiring oxygen therapy. such hospital environments are quite common in developing countries. mers cov also shares many symptoms with other respiratory diseases, consequently misdiagnosis is likely. thus the clinical staff of hospitals likely to receive returnees seeking treatment for their illnesses can act as 'sentinels' for mers cov surveillance. serious respiratory illness in the clinical staff of hospitals, especially those with icus, might be targeted for virological testing. serological testing of other staff may detect those who had asymptomatic illness. these tests would identify importation and help assess the gravity of the situation. these supplemental, limited, and targeted serological investigations would require some new financial inputs. hospital-based serum collection from selected staff is generally inexpensive and countries can manage this by themselves. storage and transportation can be tricky, but both are amenable to centralized management. health departments of developing countries can step forward to carry out this under international coordination from who. the serological tests for mers (currently available) do require specialized skill and expertise. 17 to start, it would be ideal if these tests were done under direct supervision by the labs that initially developed the tests. these labs would have to be empowered to test a large number of samples from many countries. later, the original labs might limit themselves to quality control. new funds would be required and the countries where the original labs are situated should, perhaps, come forward to support their labs to help people in the developing countries who face mers. how long will this surveillance be needed? perhaps not for long. the relevance of this surveillance strategy would end when either the outbreak extinguishes itself at the original hot spots or when clear evidence of indigenous transmission in many other sites becomes evident. the effort to optimize these in-country surveillance protocols can be managed by epidemiologists and biostatisticians of each developing country. hospital staff who are found to be immune either from asymptomatic infections or having recovered from symptomatic infection are likely to become the greatest resource when the next patient with suspected mers cov arrives. when all care givers of a patient acutely ill with mers cov are immune, further transmission within the hospital is unlikely. this way to block transmission seems a reasonable option as no vaccine is available. (in fact, this strategy can be used to manage propagated outbreaks of diseases that leave those who recover immune, like chickenpox or ebola virus disease.) outbreak management should include enhanced recruitment of recovered persons after their convalescent period for the care of new patients. the duration of immunity should be monitored as the outbreak progresses. when care givers are immune, they may provide better clinical care, as they are not afraid of contact with the patient. this approach would be a good addition to the currently recommended hospital infection control measures. 19 mers is a challenge. let us face it. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization (who) close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan missing information in animal surveillance of mers-cov lancet infect dis improving the evidence base for decision making during a pandemic: the example of 2009 influenza a/h1n1 pathogenesis of middle east respiratory syndrome coronavirus 10 years on, the world still learns from sars from sars in 2003 to h1n1 in 2009: lessons learned from taiwan in preparation for the next pandemic from sars to h7n9: the mechanism of responding to emerging communicable diseases has made great progress in china nursing shortage in india with special reference to international migration of nurses international organization for migration (iom) philippines united nations expert group meeting on international migration and development in the arab region un/pop/ egm potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus: implications for health care facilities laboratory testing for middle east respiratory syndrome coronavirus interim recommendations (revised why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? infection prevention and control during health care for probable or conformed cases of novel coronavirus (ncov) infection iype joseph, mb.bs, mph is a research scientist i (medical) in the pathogen biology group at the rajiv gandhi centre for biotechnology, kerala, india. key: cord-300536-8okuomi6 authors: baloch, zulqarnain; ma, zhongren; ji, yunpeng; ghanbari, mohsen; pan, qiuwei; aljabr, waleed title: unique challenges to control the spread of covid-19 in the middle east date: 2020-07-13 journal: j infect public health doi: 10.1016/j.jiph.2020.06.034 sha: doc_id: 300536 cord_uid: 8okuomi6 the covid-19 pandemic is spreading at unprecedented pace among the middle east and neighboring countries. this region is geographically, economically, politically, culturally and religiously a very sensitive area, which impose unique challenges for effective control of this epidemic. these challenges include compromised healthcare systems, prolonged regional conflicts and humanitarian crises, suboptimal levels of transparency and cooperation, and frequent religious gatherings. these factors are interrelated and collectively determine the response to the pandemic in this region. here, we in-depth emphasize these challenges and take a glimpse of possible solutions towards mitigating the spread of covid-19. politically, culturally and religiously a very sensitive area, which impose unique challenges for effective control of this epidemic. these challenges include compromised healthcare systems, prolonged regional conflicts and humanitarian crises, suboptimal levels of transparency and cooperation, and frequent religious gatherings. these factors are interrelated and collectively determine the response to the pandemic in this region. here, we in-depth emphasize these challenges and take a glimpse of possible solutions towards mitigating the spread of covid-19. having not forgotten the panic of the middle east respiratory syndrome coronavirus (mers-cov), its new counterpart severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of coronavirus disease 2019 (covid19) , has landed in the middle east. at june 14, 2020, there were 778147cases and 18027 deaths of covid-19 recorded across the middle east. although the case number was low cases in the early days of the outbreak, it has speeded at unprecedented pace later on and affecting all the middle east countries (figure 1 ). this could reflect the levels of preparedness and response plans implemented locally in middle east countries. the middle east lies at the juncture of eurasia and africa, and constitutes an important pillar in shaping modern civilizations and religions. it is geographically, economically, politically, culturally and religiously a very sensitive area. unfortunately, the whole region has been the center for wars and conflicts over the past years. these complexities have immense impact on breading, responding and mitigating epidemics [1] . of note, iran is the early epicenter with the highest number of confirmed covid-19 cases and deaths in the middle east region, followed by turkey, j o u r n a l p r e -p r o o f saudi arab, qatar and egypt (figure 2) . given the rapid spread of covid-19, we aim to emphasize the unique challenges of containing the epidemic in this region and to take a glimpse of possible solutions. healthcare systems within the middle east constitute a high degree of variability with regards to accessibility, capacity, and quality among different countries. the persian gulf region acquires significant wealth from the oil industry and has developed their healthcare systems [2] . within these countries, there is a two-tiered healthcare system structure with public and private streams of financing and service delivery. who has highlighted the crucial role of early detection of covid-19 cases to interrupt the spread, whereas the required expertise and diagnostic capacity could differ tremendously among different healthcare providers [3] . thus, it is essential for the authorities to ensure equal and rapid access to diagnosis and treatment of covid-19 regardless of nationalities, ethnicities, religions and beliefs. the healthcare systems in non-oil-producing countries are largely underdeveloped and were further weakened due to prolonged conflicts and wars. violent attacks on healthcare facilities and health workers during conflicts destroy essential health services that are most needed during the time [4] . the syrian conflict has been marked by frequent and intense attacks against healthcare facilities [5] . the ongoing yemen war has led to only 45% of the total healthcare facilities to be functional. a lesson learnt from the conflicts in the democratic republic of the congo is that frequently attacking health care workers and ebola treatment centers has gravely exacerbated the ebola outbreaks [6] . the geopolitical context in palestine results in limited freedom of movement and economic stability causing major challenges for healthcare system maintenance [7] . in iran, although the healthcare system has been developed, economic sanctions jeopardize social determinants of health and access to medication and healthcare [8] . the rapid growing number of covid-19 cases in iran is threating the overwhelmed healthcare system but sanctions further limit the access to emergency medical supplies. rapid and adequate access to healthcare is essential for controlling the epidemic but also for minimizing severe patient outcomes [9] . besides weakening healthcare systems, wars and conflicts in the middle east have also led to large-scale humanitarian crises. because of the syrian civil war, 13.5 out of 22 million syrian citizens require humanitarian assistance. of these, five million have been placed in refugee camps established in turkey, lebanon, jordan, egypt and other countries [10] . the conditions in yemen have continuously deteriorated since violence broke out in early 2015. over four million people have been forced to flee their homes, and more than 80% of the population (24 million) is in need of humanitarian assistance [11] . since 2016, yemen has experienced a large-scale cholera epidemic, affecting more than 1.2 million cases and over 2,500 deaths [12] . emerging evidence indicates that sars-cov-2 is very contagious [13] . conflicts-driven population migration will likely increase the risk of sars-cov-2 transmission. human-to-human transmission mainly occurs via droplets or close contacts [14] . thus the high population density in refugee camps and a lack of j o u r n a l p r e -p r o o f sanitation and hygiene are prone to widespread of the virus [15] . a subset of covid-19 patients develop diarrhea and shed viruses into feces [16, 17] . this has major implications pointing to possible fecal-oral transmission [18] . thus, it is recommended to closely monitor whether this route of transmission may occur in refugee camps where have poor water, sanitation and hygiene services. efforts must be made to minimize the risks of provoking large-scale covid-19 epidemics in the settings of humanitarian crisis. recalling the important communication lessons from the previous sars outbreak, transparent communication is key to an effective response to covid-19 outbreak. because of cultural, ethnical and religious differences, non-inclusive governments and humanitarian crises, the levels of transparent communication are clearly suboptimal in this region [19] . two factors are of concern as obstacles for transparent communication and cooperation, including religion and ethnicity oriented diversification and conflicts. mass gatherings defined by who are planned or spontaneous events that gather substantial numbers of attendees, and these events are associated with major public health challenges [20] . this is particularly relevant to the potential superspreading of sars-cov-2. the outbreak in china coincided with a massive population movement, because of the chinese lunar new year holiday [21] . a massive annual potluck banquet for 40,000 families held in wuhan in the middle of january was suspected as an exacerbation of the outbreak in the epicenter. analysis of nine gatherings for meal or holiday visit has indicated that superspreading events tend to occur during these close contacts [22] . during a religious gathering, a superspreader event that a church attendee infected nearly 40 people has occurred in korea and substantially triggered public panic. in islam, there are the five-times-daily ritual prayers and a congregational prayer on every friday in mosques, where large number of worshipers gathers. of more concerns are the large-scale pilgrimages. saudi arabia continuously receives many millions of umrah pilgrims and 2-3 million hajj pilgrims at a special season of each year from over 180 countries (fig. 3a) . respiratory infections are the most common illness among pilgrims, and the associated mortality rate during hajj has been reported as 2.4% [23] . in iran, the early covid-19 cases were recorded in qom (fig. 3b) , a city that attracts millions of pilgrims from countries including lebanon, as pilgrims concentrating on religious rituals, there are close contacts among worshipers and insufficient self-protective measures, and therefore amplify the risk of transmission and potential super spreading of sars-cov-2 [24] . the governments of saudi arabia, iran and iraq are taking heavy measures on regulating the attendance of congregational prayers and pilgrimages to mitigate the risks. in this respect, we recommend the authorities to actively communicate with religion scholars and leaders of the religious communities to facilitate decision-making and policy implementation. facing the spread of covid-19 in the middle east, there are major challenges to contain this epidemic. these include compromised healthcare systems, prolonged regional conflicts and humanitarian crises, suboptimal levels of transparency and cooperation, and frequent religious gatherings. these factors are interrelated and collectively determine the response to the epidemic in this region. there is no simple solution, but to leverage the communication and cooperation between political leaders, healthcare authorities, religion scholars and the general public is certainly important. immediate efforts should be dedicated to possibly lift economic sanctions and to end violence and conflict. for more effective response to the epidemic, we call to establish an independent and neutral international working group specifically dedicating to covid-19, with particular focus on areas with conflicts and j o u r n a l p r e -p r o o f humanitarian crises. this new organization should work closely with all united nations agencies, all the humanitarian organizations, and the local healthcare authorities with support of the international community. finally, we shall unite and ensure commitment from all parties to contain the epidemic, as there are no physical or virtual borders for sars-cov-2 to cross. all authors declare no competing interests. the work was supported by the ministry of education of china for an innovative research team in university grant (no. irt_17r88; to z.m.). armed conflicts have an impact on the spread of tuberculosis: the case of the somali regional state of ethiopia healthcare for the ageing populations of countries of middle east and north africa laboratory readiness and response for novel coronavirus (2019-ncov) in expert laboratories in 30 eu/eea countries determining the scope of attacks on health in four governorates of syria in 2016: results of a field surveillance program attacks on healthcare facilities as an indicator of violence against civilians in syria: an exploratory analysis of open-source data the exacerbation of ebola outbreaks by conflict in the democratic republic of the congo barriers preventing palestinian women from having a mammogram: a qualitative study economic sanctions threaten population health: the case of iran potential association between covid-19 mortality and health-care resource availability assessment of the health needs of syrian refugees in lebanon and syria's neighboring countries a call for urgent, organised medical missions in yemen the cholera outbreak in yemen: lessons learned and way forward a mathematical model for simulating the phase-based transmissibility of a novel coronavirus potential of large "first generation" human-to-human transmission of 2019-ncov transmission routes of 2019-ncov and controls in dental practice clinical characteristics of coronavirus disease 2019 in china epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? governance and health in the arab world mass gatherings medicine: public health issues arising from mass gathering religious and sporting events covid-19 control in china during mass population movements at new year secondary attack rate and superspreading events for sars-cov-2 mass gathering medicine (hajj pilgrimage in saudi arabia): the clinical pattern of pneumonia among pilgrims during hajj covid-19: preparing for superspreader potential among umrah pilgrims to saudi arabia key: cord-316392-bp988sir authors: kupfer, bernd; vehreschild, jörg; cornely, oliver; kaiser, rolf; plum, gerhard; viazov, sergei; franzen, caspar; tillmann, ramona-liza; simon, arne; müller, andreas; schildgen, oliver title: severe pneumonia and human bocavirus in adult date: 2006-10-17 journal: emerg infect dis doi: 10.3201/eid1210.060520 sha: doc_id: 316392 cord_uid: bp988sir nan multidrug-resistant gram-negative pathogens, and enhanced measures are needed to prevent spread of these organisms. a greater understanding of the modes of spread and acquisition of these organisms is essential for effective control of this problem. we have reported just 1 case of infection with an almost completely resistant gramnegative organism. this case expands the known geographic spread of organisms with this resistance problem. this case also underscores the importance of studying the epidemiology of antimicrobial drug resistance in gram-negative organisms in the rural setting as well as in large metropolitan centers. dissemination of knowledge regarding appropriate antimicrobial drug susceptibility testing for resistant organisms is also needed. to the editor: the newly identified human bocavirus (hbov), a member of the parvovirus family, is suspected to infect the cells of the respiratory tract and thus may be an etiologic agent of respiratory disease in humans (1) . although koch postulates have not been fulfilled for hbov, it appears likely to cause a substantial number of respiratory tract infections, at least in children (2, 3) . we describe a case of severe atypical pneumonia associated with hbov dna in a bronchoalveolar lavage (bal) sample from an adult. the patient was a 28-year-old caucasian woman with an angioimmunoblastic t-non-hodgkin lymphoma (nhl) that changed into a highly malignant blastic b-cell lymphoma (t-cell-rich b-nhl state i with 70% cd20+ cells, initial stage iiib). the patient was previously treated with vincristine and prednisone, followed by chemotherapy according to the r-choep-14 protocol (3 cycles) (november 2003 through january 2004). from january through february 2004, chemotherapy was combined with antimicrobial drug therapy according to the r-dhap protocol (which includes dexamethasone, the chemotherapy drugs cytarabine and cisplatin, and the monoclonal antibody drug rituximab) for persisting symptoms from the b-cell lymphoma. this regimen was followed by a therapy switch to alemtuzumab with ifosfamid, carboplatin, hemorrhagic cystitis occurred. during july 2004, the patient had ongoing high fever and aplasia of bone marrow with unclear etiology. on july 22, hospital treatment was initiated; it consisted of antimicrobial drug treatment with ceftriaxone (1,000 mg once daily) and gentamicin (320 mg once daily), and antimycotic therapy was started with caspofungin (50 mg once daily). since cytomegalovirus (cmv) infection was suspected, ganciclovir (250 mg twice daily) was administered iv for 2 weeks. although the patient reported an ongoing cough and pneumonialike symptoms, a severe atypical pneumonia that was refractive to antibacterial and antimycotic treatment was diagnosed for the first time during this hospital treatment. computed tomography scan showed bilateral atypical reticulonodular infiltrations predominant in the lower zones of the lungs (figure) . the bal obtained during exacerbation of the pulmonary symptoms was tested for mycobacterium tuberculosis, chlamydia pneumoniae, pneumocystis jirovecii, aspergillus sp., candida sp., cryptococcus neoformans, cmv, epstein-barr virus, hepatitis b virus, hepatitis c virus, hiv, herpes simplex virus, and vari-cella-zoster virus by pcr and culture cultivation. results were negative, except for a temporarily weak reactivity for aspergillus antigen in serum and for cmv dna in peripheral blood lymphocytes, which was positive before and became negative after ganciclovir therapy. an archived portion of the bal was assayed retrospectively by pcr/reverse transcriptase-pcr for human bocavirus, respiratory syncytial virus, human coronaviruses including severe acute respiratory syndrome-associated coronavirus, influenza virus, and human metapneumovirus (hmpv). the only positive result was obtained for human bocavirus, which was confirmed by sequence analysis of the pcr product. within a few days, the patient's symptoms decreased, and she was discharged from hospital on day 41, despite ongoing bone marrow aplasia with antimicrobial and antimycotic prophylaxis, including trimethoprim/ sulfamethoxazole (160 mg/800 mg once daily) and (voriconazole 200 mg twice daily). clinical observations led to the primary assumption that the fever, cough, and pulmonary symptoms were likely caused by the postchemotherapeutic extended bone marrow aplasia and cmv infection accompanied by an unclear bacterial but fungus-typical infection. retrospectively, however, human bocavirus dna in the archived bal strongly suggests that pulmonary symptoms were caused by this agent rather than by a yet unknown bacterial or fungal infection. thus, in the clinical episode described here, the likely causative agent responsible for the severe pneumonia was the recently described bocavirus. respiratory viruses such as respiratory syncytial virus, hmpv, and hbov seem to be the most prevalent etiologic agents of acute lower respiratory tract infection in children. recently, evidence of human bocavirus infection was reported for 3.1% to 5.7% of children <3 years of age (1) (2) (3) . previously, only limited data on adults, including immunocompromised patients, were available, but the case we describe supports the hypothesis proposed for other newly identified respiratory viruses, namely, that these pathogens also contribute to severe infections in adult patients at high risk. for example, hmpv was found in 3% of stemcell transplant recipients who underwent bal because of lower respiratory tract infection (4) . in those highrisk patients, hmpv also induced fatal infections (4) . this finding led to the conclusion that a "new" virus that induces the identical clinical symptoms, like the human bocavirus, may also contribute to severe respiratory infections. in summary, this first report of a respiratory tract infection with hbov in an adult immunocompromised patient strongly supports the assumption that hbov is an emerging pathogen that requires our attention, even for adult patients (1-3) . to the editor: leishmaniasis is a disease caused by parasites of the genus leishmania. the infection is transmitted to humans through the bites of female sandflies and manifests mainly in 3 forms: visceral, cutaneous, and mucocutaneous. visceral leishmaniasis or kala-azar, the often fatal form of the disease, is caused by species of the leishmania donovani complex. these parasites were responsible for severe recent outbreaks in sudan and other countries and are thought to originate in east africa (1) (2) (3) (4) . in this report, we describe the successful amplification of l. donovani dna in ancient egyptian and christian nubian mummies dating back 4,000 years. besides the first proof for visceral leishmaniasis in paleopathology, we provide evidence that leishmaniasis was present in nubia in the early christian period and that the organism also infected ancient egyptians, probably because of close trading contacts to nubia, during the middle kingdom. we analyzed 91 bone tissue samples from ancient egyptian mummies and skeletons and 70 bone marrow samples from naturally mummified human remains from upper nubia. the egyptian material derived from the pre-to early dynastic site of abydos (n = 7; 3500-2800 bc), a middle kingdom tomb in thebes west (42; 2050-1650 bc), and different tomb complexes in thebes west, which were built and used between the middle and new kingdom until the late period (42; c. 2050-500 bc). the nubian samples were taken before the flooding caused by the aswan dam from 2 early christian burial sites at kulubnarti, between the second and third cataracts of the nile river in northern sudan. one site was on an island in the nile and dated from 550 to 750 ad. the other was on the western bank of the nile and was in use from c.750 to 1500 ad. all samples were tested for leishmania spp. dna and further characterized by direct sequencing. in 4 of the 91 egyptian and 9 of the 70 nubian samples, a 120-bp fragment of a conserved region of the minicircle molecule of kinetoplastid mitochondrial dna of the parasite (5,6) could be successfully amplified and, with the first primer pair, unambiguously related to l. donovani species after sequencing (figure) . the positive samples from ancient egypt exclusively originated from the middle kingdom tomb, while no molecular evidence for ancient leishmania dna was found in the pre-to early dynastic and the new kingdom to late period specimens. in the middle kingdom, the egyptians extended trade relationships and military expeditions to nubia, the modern sudan, with particular interest in the gold resources of the country and in obtaining slaves to serve as servants or soldiers in the pharaoh's army. today, the sudan is one of the highly endemic countries for visceral leishmaniasis or kalaazar, which is thought to have originated in east africa and later spread to the indian subcontinent and the new world (4). therefore, the high incidence of leishmania dna in the middle kingdom samples (4 [9.5%] of 42) and the lack of findings in earlier or later time periods, may indicate that leishmaniasis was introduced into egypt at this time. leishmaniasis did not likely become endemic in the egyptian nile valley because the disease is closely linked to its vector, the phlebotomine sandfly, and the distribution of acacia-balanites woodland (7). that ancient egyptians became infected because of close trade contacts and associated travel with nubia during the middle kingdom seems more plausible. the high frequency of the opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the centers for disease control and prevention or the institutions with which the authors are affiliated. definitions of rural: a handbook for healthcare policy makers and researchers. washington: department of health and human services methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci in rural communities, western united states emergence of kpc-possessing klebsiella pneumoniae in outbreak of klebsiella pneumoniae producing a new carbapenem-hydrolyzing class a beta-lactamase, kpc-3, in a new york medical center cloning of a human parvovirus by molecular screening of respiratory tract samples evidence of human coronavirus hku1 and human bocavirus in australian children detection of human bocavirus in japanese children with lower respiratory tract infections fatal human metapneumovirus infection in stemcell transplant recipients we thank tobias allander and colleagues for the bocavirus control plasmid and carola maiworm for assistance in grammatical revision. key: cord-304054-sn7rswab authors: khan, gulfaraz; sheek-hussein, mohamud title: chapter 8 the middle east respiratory syndrome coronavirus: an emerging virus of global threat date: 2020-12-31 journal: emerging and reemerging viral pathogens doi: 10.1016/b978-0-12-819400-3.00008-9 sha: doc_id: 304054 cord_uid: sn7rswab abstract middle east respiratory syndrome (mers) is a viral respiratory illness caused by a coronavirus (cov), first identified in saudi arabia in 2012. since then, almost 2000 cases have been reported from 27 countries, with saudi arabia being the epicenter. this newly emerging virus is highly pathogenic and has a case mortality rate of 35%. it is similar to the cov causing severe acute respiratory syndrome cov (sars-cov) in that both belong to the genus beta covs that are of zoonotic origin and cause lower respiratory infection. the natural reservoir for mers-cov remains unknown. serological studies indicate that most dromedary camels in the middle east have been infected with this virus, and they maybe the potential intermediate host. however, the mode of transmission from camels to humans is poorly understood. the majority of confirmed human cases have resulted from human-to-human transmission, most probably via respiratory route. patients most at risk of developing severe mers-cov infection appear to be those with underlying conditions such as diabetes, hypertension, obesity, cardiac diseases, chronic respiratory diseases, and cancer. unlike sars-cov, mers-cov is considered an ongoing public health problem, particularly for the middle east region. in this chapter, we outline the prevailing information regarding the emergence and epidemiology of this virus, its mode of transmission and pathogenicity, its clinical features, and the potential strategies for prevention. renal failure (zaki et al., 2012; khan, 2013) . from 2012 to the end of 2017, the world health organization reported that a total of 2123 laboratories confirmed the cases of mers-cov infection and at least 740 deaths in 27 countries (case fatality rate 35%). although sizable outbreaks have been noted in several countries, the latest being in south korea (186 cases and 35 deaths) (arabi et al., 2017) , the vast number of cases ( . 80%) have been reported from saudi arabia (fig. 8.1 ) (who, 2017) . this newly emerging, highly pathogenic respiratory virus is closely related to the virus that caused an outbreak of severe acute respiratory syndrome (sars) in 2002à03. both viruses are beta covs of zoonotic origin and cause similar clinical presentations. although the natural reservoir of mers-cov infection and mode of transmission to humans is not known, one factor appears to be common to all primary cases; they are epidemiologically linked to the middle east region. most secondary cases, on the other hand, have occurred as a result of human-to-human transmission. indeed, several well-documented outbreaks have occurred in healthcare settings, often in elderly men with comorbidities (arabi et al., 2017; chafekar and fielding, 2018) . unlike sars-cov, mers-cov is an ongoing public health threat, particularly for the middle east. the fact that there is no effective antiviral drug or approved vaccine available against mers-cov makes the threat even more worrisome (zumla et al., 2016) . mers-cov is an enveloped, single-strand, and positive-sense rna virus, which belongs to the coronaviridae family. although covs are very common and can infect a variety of different animals, including cats, pigs, and bats, they rarely jump species barrier and infect humans. human covs (hcovs) were first isolated in mid-1960s, and until 2002, only two viruses, namely, hcov-229e and hcov-oc43, were known to infect humans (forni et al., 2017) . currently, six covs have been shown to infect humans. except for mers-cov and sars-cov, all others are associated with mild illnesses resembling common cold. covs are grouped into four genera, α, β, γ, and δ. the β-covs are further subgrouped in four lineages or clades, aàd (forni et al., 2017; milne-price et al., 2014) . although mers-cov and sars-cov belong to the same genus and both cause severe lower respiratory tract infection in humans, phylogenetic and sequencing data suggests that mers-cov is in fact more closely related to several bat covs (btcovs) than to sars-cov ( fig. 8 .2) (forni et al., 2017; milne-price et al., 2014) . these findings suggest that mers-cov probably is originated from a btcov ancestor (omrani et al., 2015; chan et al., 2015a,b) . the fact that covs are rna viruses exhibiting high rates of mutation and recombination, and a propensity to cross species barrier, increases the risk of new variants emerging with higher virulence and transmission sabir et al., 2015) . the replication cycle of mers-cov consists of a number of important steps: attachment and entry into host cell, uncoating and release of viral rna, transcription and translation of viral specific genes, replication of viral genomic rna, and assembly and release of progeny virions from the infected cell. as is typical of most rna viruses, all of these steps take place in the cytoplasm of the host cell (de wit et al., 2016) . the initial attachment of mers-cov to its susceptible host cells is mediated by the viral envelop spike glycoprotein s binding to its cellular receptor, cd26 (also known as dipeptidyl peptidase 4, dpp4) (lu et al., 2013; raj et al., 2013) . a number of different cell types express dpp4 and hence are susceptible to mers-cov infection including pneumocytes, alveolar macrophages, bronchial epithelia, vascular endothelium, as well as a subset of mononuclear cells (meyerholz et al., 2016; yu et al., 2017) . following attachment, the virus enters the susceptible cell by fusion of its envelope with the plasma membrane and/or via receptor-mediated endocytosis (de wit et al., 2016) . once in the cytoplasm of the target cell, the virus particle uncoats and the positive-sense viral rna binds to ribosomes, and the viral rna-dependent rna polymerase is translated. this enzyme in turn transcribes full-length negative-sense rna that forms the template for the production of positive-sense viral genome. the viral polymerase also generates various individual mrnas that are translated into viral proteins. viral structural proteins and viral genomic rna are assembled into new virus particles in the rough endoplasmic reticulum-golgi intermediate compartment and eventually released out of the cell by exocytosis. from the infected host, it appears that the virus is shed in nasal secretions (adney et al., 2014) . interestingly, in bats, a recent study revealed that dpp4 receptor is rarely expressed in epithelial cells of respiratory tract, but highly expressed in epithelial cells of intestinal tract, indicating that fecalàoral is probably the main mode of transmission in bats (widagdo et al., 2017) . of all the documented cases to date, there is no evidence for the transmission of the virus from bats or their droppings directly to humans. we also have limited data on the survival of the virus outside its host. when the virus was added to milk from dromedary camels, goats, or cow and stored at 4 c or 22 c, the virus could be recovered up to 72 and 48 hours, respectively (van doremalen et al., 2013) . pasteurization of the milk, however, completely destroyed mers-cov infectivity (van doremalen et al., 2013) (table 8 .1). the current prevalent view is that mers-cov is a zoonotic virus that entered the human population in the arabian peninsula, via direct or indirect contact with infected dromedary camels. studies indicate that the virus had been circulating in the camel population for decades, and only recently "jumped" the species barrier to infect humans. what are the factors that precipitated the virus to cross the species barrier are unknown. most of the confirmed cases of mers-cov infection in humans have been via person-to-person transmission. the epidemiological elements in the transmission of mers-cov appear to be factors related to the virus, the host, and the environment. cases have occurred as sporadic infections, family clusters, or outbreaks in healthcare settings (kim et al., 2017; oboho et al., 2015) . although the infection is limited and nonsustained, outbreaks in healthcare settings have been particularly extensive and worrisome. the nonspecific initial symptoms, late diagnosis, and inadequate infection control measures have all contributed to the outbreaks in healthcare settings (oboho et al., 2015; hunter et al., 2016; kim et al., 2017) . although mers-cov cases have been detected in many countries around the world, almost all have been directly or indirectly linked to the middle east region (table 8 .2). one of the most notable outbreaks outside the middle east occurred in south korea in may 2015 (kim et al., 2017; lee and wong, 2015) . a single infected man returning from the middle east caused a hospital outbreak in which 185 individuals were infected (kim et al., 2017) . the epidemiological pattern observed in the korean outbreak was similar to that observed in the middle east; more males than females were affected, most of the 38 patients who died had underlying conditions such as respiratory disorders, cancer, hypertension, cardiovascular problems, or diabetes (kim et al., 2017) . it is noteworthy that the death rate was lower in the cases from south korea compared to those reported from saudi arabia (23% vs 47%) (virlogeux et al., 2016) . the reason for this is not clear. although more than 80% of mers-cov cases have occurred in saudi arabia, the virus clearly has the potential of spreading to other countries. thus there is an obvious need to detect, respond, and contain any outbreak of mers-cov cases if we want to prevent the global spread of the virus. unfortunately, this is easier said than done. there are a number of risk factors prevalent in some of the countries of the middle east which support the emergence and reemergence of infectious diseases (buliva et al., 2017) . these risk factors include political instability, famine and war, less developed healthcare infrastructure, weak public health and surveillance systems, increased population growth and mobility, climate change, and urbanization (buliva et al., 2017) . in order to prevent the emergence and spread of infectious diseases such as mers-cov, it is essential to address the underlying causes and risk factors. needless to say, these are major challenges for any country, let alone the eastern mediterranean region. to successfully address these challenges, it will require not only funding, establishment of robust and effective surveillance systems, and national and international corporations but also above all, peace and security in the region. infection with mers-cov, in its initial description, resembled "sars-like" illness (chan et al., 2015a,b) . further analysis of the epidemiological, virological, and clinical aspects of mers-cov and sars-cov revealed important differences between the two viruses. identifying unique aspects of mers-cov helped to explain how the epidemic evolved and the steps that could be taken to prevent its spread (chan et al., 2015a,b) . serological studies have indicated that most dromedary camels in africa and the middle east, but not other animals such as sheep, goats, and cows, were seropositive for mers-cov . moreover, seroprevalence in dromedary camels appears to vary, with high rates reported in animals from egypt, ethiopia, nigeria, and sudan and lower rates in animals from tunisia (ali et al., 2017) . intriguingly, dromedaries from australia, canada, the united states, germany, netherlands, and japan have been reported to be seronegative for mers-cov (omrani et al., 2015) . importantly, population-based seroepidemiologic studies indicated that the seroprevalence of the virus was several folds higher in people who have been exposed to camels compared to those in the general population ( accumulating serologic and molecular evidence indicates that the virus in dromedaries is genetically similar to mers-cov in humans, supporting the notion that dromedary camels could be the potential source of infection to human memish et al., 2014a,b,c; sabir et al., 2015) . indeed, mers-cov antibodies have been isolated in dromedary camels across the arabian peninsula, north africa, and eastern africa dating from as far back as the 1990s (milne-price et al., 2014; omrani et al., 2015) . this finding suggests that mers-cov may have been circulating in dromedaries for over 20 years before it was first recognized as a cause of human infection (aly et al., 2017) . in a recent study, a fatal case of mers-cov infection was reported in an individual who had direct contact with a dromedary camel (azhar et al., 2014) . sequence analysis of the virus isolated from the case and the camel was identical, clearly indicating that mers-cov can indeed be transmitted from camels to human (azhar et al., 2014) . it appears that active infection with release of the virus in nasal secretions, particularly during the incubation period, is important for the transmission of the virus to humans . where and how the camels acquired the infection remains unknown. it has been hypothesized that bats could be the potential source (fig. 8.3 ) (anthony et al., 2017; mohd et al., 2016; omrani et al., 2015) . indeed, mers-cov-like viruses have been identified in certain species of bats (anthony et al., 2017; woo et al., 2006) . the bats are present in most parts of the world and often infected with various zoonotic viruses. thus it is plausible that at some point in the past, camels acquired the infection from bats, leading to a sustained infection in the camel population (fig. 8.3) . mers-cov rna has been identified in the milk, nasal secretion, and feces of dromedary camels (omrani et al., 2015) . since camels and humans are often in close contact, particularly in the arab gulf states, humans would be at increased risk of contracting the virus from actively infected animals (mackay and arden, 2015; reusken et al., 2015) . indeed, mers-cov seropositivity in shepherds and those working in slaughterhouses in saudi arabia has been reported to be an order of magnitude higher than in the general population (arabi et al., 2017) . although possible, no evidence currently exists to support the transmission of mers-cov from bats to humans directly. what is certain is that transmission of the virus can occur from camels to humans, but the process is still not fully understood (al hammadi et al., 2015; memish et al., 2014a,b,c) . one possibility is that some species of covs from camels and humans could recombine leading to the emergence of a new virus that can infect both, camels and human (sabir et al., 2015) . most mers-cov infections in humans occur through human-tohuman contact (arabi et al., 2017; zumla et al., 2016) . available data on epidemiologic observations suggest that human-to-human transmission occurs primarily through close contact with an infected individual. the mode of transmission is presumed to be via respiratory droplets or aerosols, with higher risk in situations where aerosols are generated, and inadequate personal protection or proper room ventilation is not present (kutter et al., 2018) . in the south korean outbreak a total of 185 individuals were infected; 136 of whom were directly infected by just 3 cases, the so-called super spreaders. late diagnosis, lack of infection control measures, poor communication and healthcare management procedures, and failure to quarantine the "super spreaders" were identified as major factors contributing to this large nosocomial outbreak (kim et al., 2017) . cov is a common cause of mild respiratory tract infection manifesting as common cold. it is estimated that approximately one-third of all upper respiratory tract infections in adults are due to covs. sars-cov and mers-cov are the exceptions. both of these viruses have a high propensity to infect the lower respiratory track and lead to severe disease and death (de wit et al., 2016) . the finding that both of these viruses, but in particular, mers-cov, are able to evade the body's immune responses and infect a broad range of cells, explaining the widespread infection and development of severe disease (mackay and arden, 2015) . it is noteworthy that, even in the absence of viral shedding in the upper respiratory tract, most symptomatic patients have abnormal chest radiographs (fig. 8.4) (assiri et al., 2013; de wit et al., 2016) . the incubation period for mers-cov infection is about 5à6 days with most patients showing symptoms within 14 days of exposure (de wit et al., 2016; virlogeux et al., 2016) . the initial clinical symptoms of mers-cov infection can range from asymptomatic to low-grade fever, cough, sore throat, myalgia, and less frequently diarrhea and vomiting. progression to more severe disease is characterized by the symptoms of shortness of breath, severe pneumonia, respiratory distress syndrome, multiorgan failure, and death (arabi et al., 2017; de wit et al., 2016; guery et al., 2013) . the severity of the infection appears to vary depending on the age of the patient and any underlying conditions. adults over the age of 50 years and with comorbidities such as diabetes, hypertension, chronic renal or lung disease, cancer, and heart disease are at increased risk of developing severe disease and death (badawi and ryoo, 2016) . although the vast majority of confirmed cases have been in male adults, children are also susceptible to infection, albeit at lower rate and with milder disease . based on limited data, mers-cov infection in pregnancy can also lead to maternal and perinatal disease and death assiri et al., 2016) . not surprisingly, the severity of infection and the risk of transmission of mers-cov are significantly increased in environments such as hospitals (cho et al., 2016; hastings et al., 2016) . the clinical symptoms of mers-cov infection, especially in early stages of the infection, are typically nonspecific and can resemble a number of acute respiratory tract infections. however, acute febrile respiratory illness in a patient with a recent travel history to the middle east or direct/indirect contract with a confirmed case of mers-cov should be enough suspicion to request laboratory testing for mers-cov. studies indicate that viral replication and shedding is higher in lower compared to upper respiratory tract (de wit et al., 2016; memish et al., 2014a,b,c) . hence, for laboratory diagnosis, lower respiratory tract specimens such as tracheal aspirate, bronchoalveolar lavage, or pleural fluid are preferred over upper respiratory tract specimens such as nasopharyngeal swab (mackay and arden, 2015; memish, et al., 2014a,b,c) . it is essential that appropriate personal protective equipment (ppe) and infection control measures are implemented when dealing with suspected cases. the assay of choice for the laboratory diagnosis of mers-cov is reverse transcriptase real-time polymerase chain reaction (rt-pcr) on respiratory samples. this assay was established soon after the identification of mers-cov back in 2012 (zaki et al., 2012) . rt-pcr is not only very sensitive but also importantly a fairly rapid technique, which is essential for early diagnosis and quarantine implementation. the virus can also be cultured. a number of different cell lines are susceptible for in vitro infection, including vero and llc-mk2 cells (zaki et al., 2012) . however, cell culture approach is very slow and not easily adaptable to every diagnostic laboratory, hence the preference of rt-pcr. for determining past infection or for surveillance studies, the detection of antibodies to mers-cov using serological assay, such as enzyme linked immunosorbent assay (elisa), can be performed (mackay and arden, 2015) . currently, no specific antiviral therapy or vaccine is available for the treatment and prevention of mers-cov infection. supportive care and prevention of complications are the main management options that are available. however, efforts are underway for the development of therapeutic and vaccine candidates. in a marmoset model of mers-cov infection, several compounds, including ribavirin, lopinavir/ritonavir, interferon-β1b, and interferon-α2b, alone or in combinations, have shown varying degree of success (chan et al., 2015a,b; falzarano et al., 2013) . similarly, passive immunotherapy with neutralizing antibodies against mers-cov has also shown some therapeutic value in inhibiting viral replication (luke et al., 2016) . in terms of vaccines, several potential candidates have been developed and are in different stages of clinical testing (du et al., 2016) . the possibility of developing an effective vaccine based on mers-cov spike protein is promising . in the absence of licensed antiviral or vaccine, current strategies of combating mers-cov infection are aimed at reducing the risk of animal-to-human and human-to-human transmissions. strategies recommended include avoidance of drinking unpasteurized camel milk, limiting direct contact with a sick animal, avoidance of close contact, and sharing of utensils with an infected individual, using ppe when in direct contact with an infected person and proper hand hygiene. in addition, early recognition and laboratory confirmation of infected cases, segregation/isolation of infected cases, and contact tracing and strict implementation of infection control measures in healthcare settings are all essential for controlling and preventing of mers-cov infection and spread. replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from 2012 to 2015 asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia evidence for camel-to-human transmission of mers coronavirus prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives mers-cov: understanding the latest human coronavirus threat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study sars and mers: recent insights into emerging coronaviruses transmission of mers-coronavirus in household contacts vaccines for the prevention against the threat of mers-cov treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques molecular evolution of human coronavirus genomes the spike-protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2 and is targeted by neutralizing antibodies clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels outbreak of middle east respiratory syndrome at tertiary care hospital transmission of middle east respiratory syndrome coronavirus infections in healthcare settings a novel coronavirus capable of lethal human infections: an emerging picture middle east respiratory syndrome infection control and prevention guideline for healthcare facilities middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications transmission routes of respiratory viruses among humans probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo mers coronavirus: diagnostics, epidemiology and transmission screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome human infection with mers coronavirus after exposure to infected camels, saudi arabia jumping species-a mechanism for coronavirus persistence and survival dipeptidyl peptidase 4 distribution in the human respiratory tract the emergence of the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: references emerging and reemerging viral pathogens a nationwide, cross-sectional, serological study mers-cov outbreak in jeddah-a link to health care facilities middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction evidence of person-to-person transmission within a family cluster of novel coronavirus infections dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study occupational exposure to dromedaries and risk for mers-cov infection co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia confirmed global cases of mers-cov mers-cov global summary and assessment of risk tissue distribution of the mers-coronavirus receptor in bats molecular diversity of coronaviruses in bats comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to sars-coronavirus coronaviruses-drug discovery and therapeutic options key: cord-252222-wyamc46k authors: leung, chi hung czarina; gomersall, charles david title: middle east respiratory syndrome date: 2014-05-13 journal: intensive care med doi: 10.1007/s00134-014-3303-y sha: doc_id: 252222 cord_uid: wyamc46k nan middle east respiratory syndrome (mers) is due to rna betacoronavirus (mers-cov) infection. by february 2014, the world health organization (who) had received reports of 181 laboratory-confirmed cases in the middle east, europe and northern africa, with all having connections to the middle east. the median age is 52 years, with male predominance (64.5 %) [1] . severe cases deteriorate rapidly: median time from symptom onset to icu admission is 2 days and to intubation is 4.5-6 days, with an icu admission rate of 51.6 % [2, 3]. mortality amongst critically ill ventilated patients is high with a 28-day mortality of 42 % and a 90-day mortality of 58 %. the median icu stay is 30 days [2] . the apparent epidemiology may be biased by selective reporting of more severe cases and the small total number of patients makes it susceptible to distortion by individual outbreaks. for example, 23 of the 181 cases were in a single outbreak involving three healthcare facilities, including a haemodialysis unit [4] , and more than half of all secondary cases have been nosocomial [5], increasing the prevalence of co-morbidity (75.8 %). bats [6] and camels [7] have been implicated as primary animal hosts; however, the data are not conclusive. human to human transmission occurs with a relatively low basic reproductive number (r 0 ) of 0.42-1.5 [8, 9] and an incubation period of approximately 5 days [3, 10] but up to 15 days [4] . the mode of transmission is unknown, but relatively simple infection control measures effectively controlled a nosocomial outbreak [4]. in an ex vivo model mers-cov rapidly achieved a high viral load, infecting type i and ii alveolar cells. cell entry appears to be via proline exopeptidase (dpp4) receptors, which are expressed in lung and kidney. histological changes include detachment of type ii cells from basement membrane, disruption of alveolar tight junctions and changes consistent with apoptosis [10] . viral nucleic acid in patients' faeces and urine suggests direct involvement of gastrointestinal and urinary tracts [11] . the presentation is non-specific with fever, chills, sore throat, myalgia, arthralgia and dyspnoea. vomiting and diarrhoea are common. chest x-ray changes are consistent with viral pneumonitis and acute respiratory distress syndrome. clinical suspicion, therefore, depends on vigilance and, for the present time, on a history of travel to the middle east or contact with a patient with respiratory disease and an appropriate travel history [11] . the prevalence of organ failure is difficult to determine owing to reporting of limited icu data and possible duplicate reporting of cases [2, 4]. however, almost all icu admissions required mechanical ventilation [2] [3] [4] 11] . a recent case series reported 11 critically ill patients with confirmed mers and one with probable mers [2]. all required invasive mechanical ventilation. the median (range) p/f ratio on day 1 was 88 (76-413). six patients required high frequency oscillation, nitric oxide or prone ventilation with four requiring a combination. median duration of ventilation was 16 days. five received noninvasive ventilation but all progressed to invasive ventilation, in keeping with other reports [4, 11, 12] . acute kidney injury was common, with 7/12 critically ill patients requiring renal replacement therapy [2], consistent with other reports [11, 12] . shock developed in 11/12 patients and appeared to be moderately severe [2]. routine investigations are neither specific nor sensitive. lymphopenia and thrombocytopaenia occur in about onethird of patients, but approximately 10 % have lymphocytosis. lactate dehydrogenase is raised in about half and hepatic transaminases in 10-15 % [3]. lower respiratory tract specimens (preferably, due to higher viral load) and oropharyngeal and nasopharyngeal samples should be obtained [11] . viral shedding may vary with time and repeated sampling is recommended for patients with high suspicion of mers-cov infection, even if initial results are negative [11] or other organisms are identified [2]. blood, urine and stool specimens tested by real-time reverse-transcription polymerase chain reaction for mers-cov rna targets, along with conjunctival swabs and cerebral spinal fluid if conjunctivitis or encephalitis are suspected. there is no effective disease-specific treatment or vaccine. early ribavirin and interferon reduced severity of respiratory symptoms, radiology, inflammatory markers and viral load in rhesus macaque monkeys but was ineffective in humans with mers and ards. however this may be due to late administration and severity of disease [13] . there is no evidence of benefit from high dose steroid [1] and sars data indicate a significant risk of harm [14] . a management algorithm is given in fig. 1 . currently, the epidemic or pandemic risk appears to low. the estimated r 0 (the number of secondary infections generated by a primary infection in a susceptible population) ranges from 0.42 to 1.5. if this number is less than 1, then transmission is guaranteed to fade away. if greater than 1, there is a risk of an epidemic; but if it is only a little over 1 (i.e. 1-1.5), then transmission may fade away anyway [15] . however, there are a number of caveats. firstly, r 0 is difficult to estimate [15] . secondly, coronaviruses rapidly adapt to new hosts and in the process may become more infectious. thirdly, the existence of mild, perhaps unidentified, cases makes infection control measures less likely to be effective. approximately 60 % of mers cases may have been undiagnosed [8] . fourthly, a significant epidemic will pose a major challenge to intensive care, owing to the prolonged duration of mechanical ventilation and high requirement for renal replacement therapy. non-invasive ventilation does not appear to be useful, except possibly to delay intubation, and may increase disease transmission and risk undue delay in intubation: four patients who received cpap suffered six cardiac arrests [4]. as the mode of transmission is unknown implement airborne and contact precautions until proved unnecessary [11] . personal protective equipment against airborne transmission includes a fit-tested ffp2 (or equivalent) face mask. fit testing is time-consuming and should be carried out in advance of hospital admission of a patient with mers as the delay between onset of symptoms and icu admission [2] is very short. visiting without full personal protective equipment has been associated with nosocomial transmission [11] and visitors should also be fit-tested. middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs middle east respiratory syndrome: new disease, old lessons clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids assessing the pandemic potential of mers-cov key: cord-269232-rhhmvnlp authors: joseph, sunitha; wernery, ulrich; teng, jade ll; wernery, renate; huang, yi; patteril, nissy ag; chan, kwok-hung; elizabeth, shyna k; fan, rachel yy; lau, susanna kp; kinne, jörg; woo, patrick cy title: first isolation of west nile virus from a dromedary camel date: 2016-06-08 journal: emerg microbes infect doi: 10.1038/emi.2016.53 sha: doc_id: 269232 cord_uid: rhhmvnlp although antibodies against west nile virus (wnv) have been detected in the sera of dromedaries in the middle east, north africa and spain, no wnv has been isolated or amplified from dromedary or bactrian camels. in this study, wnv was isolated from vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in dubai. complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. there was no clustering of the present wnv with other wnvs isolated in other parts of the middle east. within lineage 1a, the dromedary wnv occupied a unique position, although it was most closely related to other wnvs of cluster 2. comparative analysis revealed that the putative e protein encoded by the genome possessed the original wnv e protein glycosylation motif nys at e154–156, which contained the n-linked glycosylation site at n-154 associated with increased wnv pathogenicity and neuroinvasiveness. in the putative ns1 protein, the a70s substitution observed in other cluster 2 wnvs and p250, which has been implicated in neuroinvasiveness, were present. in addition, the foo motif in the putative ns2a protein, which has been implicated in neuroinvasiveness, was detected. notably, the amino-acid residues at 14 positions in the present dromedary wnv genome differed from those in most of the closely related wnv strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative e and ns5 proteins. the present study is the first to demonstrate the isolation of wnv from dromedaries. this finding expands the possible reservoirs of wnv and sources of wnv infection. west nile virus (wnv) is a positive-sense single-stranded ribonucleic acid (rna) virus in the flaviviridae family. wnv is the leading cause of mosquito-borne encephalitis in humans in many parts of the world. in addition to humans, many animals are also susceptible to wnv infections. large outbreaks have occurred globally in both humans and other animals, such as horses and pigs. birds are the natural reservoirs of wnv. bird-to-bird, bird-to-mammal and bird-to-human transmissions are achieved by mosquito bites, with humans and other mammals serving as dead-end hosts because of low viral loads. camels are one of the most unique mammals on earth and have shown perfect adaptation to desert life. there are two surviving old-world camel species: camelus dromedarius (dromedary or onehumped camel), which inhabits the middle east and north and northeast africa, and camelus bactrianus (bactrian or two-humped camel), which inhabits central asia. among the 20 million camels on earth, 90% are dromedaries. although wnv is known to infect some of the new world camels, such as llamas and alpacas, 1 only antibodies against wnv have been detected in the sera of old-world camels, such as the dromedaries in the middle east, north africa and spain, with a seroprevalence of 3%-38%. [2] [3] [4] [5] to date, no wnv has been isolated or amplified from either dromedary or bactrian camels. recently, the emergence of middle east respiratory syndrome (mers) and the isolation of the mers coronavirus (mers-cov) from dromedaries boosted interest in the search for novel viruses in dromedaries. [6] [7] [8] [9] [10] [11] [12] [13] in this article, we report the first isolation of wnv from a dromedary calf in the united arab emirates during the process of mers-cov screening and the results of the comparative genome and phylogenetic analysis. clinical samples were obtained during a necropsy of a dromedary calf at the central veterinary research laboratory in dubai, the united arab emirates, using standard procedures. the central veterinary research laboratory in dubai is the center for performing necropsies of dromedaries from sheikhs in the uae with the aim of finding the cause of death and preventing the spread of infectious diseases to other camels or herds. nasal swabs and pooled ground trachea/lung samples were inoculated onto vero cells for mers-cov screening. 14 the pooled clinical samples were diluted 10-fold with viral transport medium and filtered. two hundred microliters of the filtrate was inoculated into 200 μl of minimum essential medium (gibco, grand island, ny, usa). four hundred microliters of the mixture was added to 24-well tissue culture plates with vero cells by adsorption inoculation. after 1 h of adsorption, the excess inoculum was discarded, the wells were washed twice with phosphate-buffered saline, and the medium was replaced with 1 ml of minimum essential medium (gibco). cultures were incubated at 37°c with 5% co 2 and inspected for cytopathic effects daily using inverted microscopy. negative-contrast electron microscopy was performed as previously described. 15 tissue culture cell extracts infected with dromedary wnv were centrifuged at 19 000g at 4°c. then, the pellet was resuspended in phosphate-buffered saline and stained with 2% phosphotungstic acid. samples were examined with a philips em208s electron microscope (philips scientifics, eindhoven, the netherlands). sample preparation for illumina sequencing rna was extracted from the isolated virus using the qiaamp viral rna mini kit (qiagen, hilden, germany). reverse transcription and polymerase chain reaction (pcr) were performed using the super-script iii reverse transcriptase (invitrogen, carlsbad, ca, usa) and a random primer containing a 20-base arbitrary sequence at the 5′ end followed by a randomized octamer (8n) at the 3′ end. a single round of priming and extension was performed using the klenow fragment polymerase (new england biolabs, ipswich, uk). pcr amplification with a primer consisting of only the 20-base arbitrary sequence of the random primer was performed with 20 cycles of 94°c for 15 s, 60°c for 30 s and 68°c for 1 min and a final extension at 68°c for 7 min in an automated thermal cycler (applied biosystems, carlsbad, ca, usa). standard precautions were taken to avoid pcr contamination, and no amplified pcr product was observed in the negative control. the pcr product was purified using the minelute pcr purification kit (qiagen) following the manufacturer's protocol. the purified dna was eluted in 15 μl of eb buffer and used as the template for library construction. 16 library construction for illumina sequencing the metagenomics library was generated using a dna library prepared with the nextera xt dna sample preparation kit (illumina, san diego, ca, usa) according to the manufacturer's protocol. briefly, 1 ng of input dna was tagmented by the nextera xt transposome at 55°c for 5 min. this transposome simultaneously fragmented the input dna and added an adapter sequence to the ends, thereby allowing amplification by pcr in subsequent steps. the sequencing library with the tagmented dna was amplified in 12 cycles of 95°c for 10 s, 55°c for 30 s and 72°c for 30 s and a final extension at 72°c for 5 min in an automated thermal cycler (applied biosystems). the amplified dna library was purified using 1.8 × ampure xp beads (beckman coulter, indianapolis, in, usa) to remove very short library fragments from the population. the amplified dna library was analyzed using the 2100 bioanalyzer instrument (agilent technologies, santa clara, ca, usa). the purified sequencing library was quantified using the kapa library quantification kit (kapa biosystems, wilmington, de, usa) and subsequently sequenced on the illumina hiseq 2500 with 151 bp paired-end reads (rapid run mode). image analysis and base calling were performed with scs2.8/rta1.8 (illumina). fastq file generation and the removal of failed reads were performed using casava ver.1.8.2 (illumina). 16 genome assembly and complete-genome sequencing illumina sequence reads were quality trimmed by prinseq-lite, and de novo genome assembly was performed with mira 4.9. the 5′ and 3 0 ends of the viral genome were confirmed by rapid amplification of cdna ends (race) using the 5′/3′ race kit (roche, penzberg, germany). the nucleotide sequence of the genome and the deduced amino-acid sequences of the open reading frames were compared with those of other wnv strains with complete genomes in the vipr sequence database (http://www.viprbrc.org/). n-linked glycosylation sites were predicted using the netnglyc 1.0 software (http://www.cbs.dtu.dk/ services/netnglyc/). complete polyprotein nucleotide sequences were aligned with muscle. a maximum-likelihood phylogenetic tree with 1000 bootstraps was constructed using the gtr substitution model; gamma distribution among sites was conducted in mega5. the nucleotide sequence of the dromedary wnv genome isolated in this study has been lodged in the genbank sequence database under accession no ku588135. necropsy of the female 1-month-old 50 kg fresh dromedary calf revealed pale muscles and myocard, massive lung congestion and colitis. the causes of death were white muscle disease, colisepticemia and candida enteritis. at the first passage, the vero cells inoculated with both the nasal swab and pooled trachea/lung samples showed identical cytopathic effects on day 4, with cell rounding, progressive degeneration and detachment ( figure 1a) . electron microscopy showed enveloped icosahedral virions 45-50 nm in diameter with a 25-to 35-nm electron dense core ( figure 1b ). both the cytopathic effects and viral morphology were inconsistent with those of mers-cov. the complete genome of the isolated virus was sequenced and assembled. the size, g+c content and genome structure were similar to other wnvs. the single polyprotein is putatively cleaved by proteases into three structural proteins (capsid (c), premembrane/ membrane (prm/m) and envelope (e)) and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5). phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a (figure 2a ). 17 the present dromedary wnv occupied a unique position within lineage 1a, although it was most closely related to other wnvs of cluster 2 ( figure 2b ). comparative analysis based on the complete polyprotein sequence showed the highest (99.51%-99.56%) overall amino-acid identities to wnv strains isolated from a mosquito in kenya (genbank accession number ay262283), horses in italy and morocco (genbank accession numbers af404757, ay701413 and ay701412) and a human in italy (genbank accession number jq928175). detailed annotation revealed that the putative e protein encoded by the genome possessed the original wnv e protein glycosylation motif nys at e154-156, which contained the n-linked glycosylation site at n-154 associated with increased wnv pathogenicity and neuroinvasiveness. 18 in the putative ns1 protein, the a70s substitution observed in other cluster 2 wnvs and p250, which was implicated in neuroinvasiveness, were also present. 19 in addition, the foo (flavivirus overlapping orf) motif in the putative ns2a protein that was implicated in neuroinvasiveness was detected. 20 notably, 14 amino-acid residues in the present dromedary wnv genome differed from those in most of the closely related wnv strains in cluster 2 of lineage 1a (figure 3) , with the majority of these differences observed in the putative e and ns5 proteins. we report the first isolation of wnv in a dromedary. the first evidence for the presence of hemagglutination inhibiting and complement-fixing antibodies in dromedaries from nigeria against wnv was published in 1990. 21, 22 recently, a study has shown the presence of neutralizing antibodies against wnv from dromedaries in north africa. 3 in the united arab emirates, antibodies against wnv have been reported in 38% of 1119 dromedary sera 5 and 19.2% of 750 tested equine sera, 23 supporting the conclusion that wnv is present in the country. in the present study, we isolated a wnv from two independent respiratory samples from a dromedary calf using vero cells, which is one of the most widely used cell lines for the recovery of wnv. this finding was consistent with previous studies that showed that wnv could also be detected in respiratory specimens from birds, humans and other mammals. 24, 25 notably, the glycosylation motif nys in the e protein, the a70s substitution and p250 in the ns1 protein and the foo motif in the ns2a protein, which are important for pathogenicity and neuroinvasiveness, were also detected in the present wnv isolate. [18] [19] [20] however, because there were no clinical signs in the dromedary calf, the pathogenic role of wnv in dromedaries remains to be determined. viral cultures using other samples from the dromedary were not performed because the samples were initially intended for mers-cov isolation. the present dromedary wnv belongs to wnv lineage 1a. the whole-genome phylogenetic trees showed that the present dromedary wnv was most closely related to other wnvs in cluster 2 of lineage 1a with high bootstrap support ( figure 2 ). however, there was no clustering of the present wnv with wnvs isolated in other parts of the middle east, such as israel, egypt and cyprus (data not shown). indeed, two wnvs from israel (genbank accession number ay688948) and cyprus (genbank accession number gq903680) belong to lineage 2, another isolate from israel (genbank accession number hm152773) belongs to cluster 4 of lineage 1a, and two other isolates from israel (genbank accession number hm051416) and egypt (genbank accession number eu081844) belong to cluster 1 of lineage 1a. moreover, 14 amino-acid positions located in various regions of the genome encoding five different proteins showed marked differences with the corresponding amino acids in the genomes of the isolate's close relatives in cluster 2. interestingly, most of the amino-acid differences between the present dromedary wnv and the closely related wnvs from africa and europe are located in the e and ns5 genes. the e protein is generally the least conserved protein because it is exposed to external selection pressure, whereas the ns5 protein is highly conserved because it contains the viral methyltransferase and rna-dependent rna polymerase. complete-genome sequencing of more wnv strains and comparative genomic and phylogenetic studies are needed to ascertain whether dromedary wnvs form a unique cluster in lineage 1a. the mers epidemic and the discovery of dromedaries as the reservoir of mers-cov have boosted interest in the search for novel viruses in dromedaries. prior to 2013, viruses from at least eight families were found to infect camels. in the last two years, we have reported the discovery of a novel dromedary camel cov (uae-hku23), a novel genotype of hepatitis e virus, a novel genus of enterovirus, a novel astrovirus, and novel picobirnaviruses and circoviruses in dromedaries. 16, [26] [27] [28] [29] these discoveries have remarkably widened the spectrum of viruses known to infect dromedaries. notably, after the description of the novel genotype of hepatitis e virus in dromedaries, another group found this dromedary camel hepatitis e virus as a cause of chronic hepatitis e infection in a liver transplant recipient with a habit of camel milk and meat consumption, highlighting the importance of camels as a source of infectious diseases. 30 the present study is the first demonstration of the isolation of wnv in dromedaries and the first wnv complete-genome sequenced from a dromedary among the relatively few full-length wnv genome sequences from the middle east. further studies will help elucidate whether dromedaries can serve as another possible source of wnv infection and determine the tissue tropism of wnv in s joseph et al figure 3 summary of amino-acid changes in the various putative proteins encoded by the dromedary wnv genome relative to those of its most closely related wnv strains in cluster 2 of lineage 1a. only changes that occurred in ⩾ 3 strains are shown. dots indicate no difference from the dromedary wnv. west nile virus in a dromedary s joseph et al viral diseases of new world camelids a transversal study on antibodies against selected pathogens in dromedary camels in the canary islands rift valley and west nile virus antibodies in camels emerging viral diseases in dromedary camels in the southern morocco seroepidemiological studies for the detection of antibodies against nine infectious diseases in dairy dromedaries (part-1) middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of a novel coronavirus from a man with pneumonia in saudi arabia a phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease acute middle east respiratory syndrome coronavirus infection in livestock dromedaries isolation and characterization of a novel betacoronavirus subgroup. a coronavirus, rabbit coronavirus hku14, from domestic rabbits metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses the challenge of west nile virus in europe: knowledge gaps and research priorities viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the new york strain of west nile virus loss of dimerisation of the nonstructural protein ns1 of kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of ns1 ns1' of flaviviruses in the japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness a survey for haemagglutination-inhibiting antibody to west nile virus in human and animal sera in nigeria west nile complement fixing antibodies in nigerian domestic animals and humans west nile fever in the united arab emirates west nile virus detection in kidney, cloacal, and nasopharyngeal specimens fatal hemorrhagic fever caused by west nile virus in the united states novel betacoronavirus in dromedaries of the middle east new hepatitis e virus genotype in camels, the middle east a novel astrovirus from dromedaries in the middle east a novel dromedary camel enterovirus in the family picornaviridae from dromedaries in the middle east chronic infection with camelid hepatitis e virus in a livertransplant recipient who regularly consumes camel meat and milk we are grateful to dr ismail hassab elrasoul mohammed khair (central veterinary research laboratory, dubai, uae) for sending the carcass. we also thank the members of the centre for genomic sciences, the university of hong kong, for their technical support. this work is partially supported by the theme-based research scheme (project no t11/707/15), the university grant committee, and the strategic research theme fund, the university development fund, the university of hong kong. dromedaries. other studies on the epidemiology, disease spectrum and ecology of wnv in this group of unique animals are also warranted. key: cord-275602-cog4nma0 authors: watkins, kevin title: emerging infectious diseases: a review date: 2018-06-22 journal: curr emerg hosp med rep doi: 10.1007/s40138-018-0162-9 sha: doc_id: 275602 cord_uid: cog4nma0 purpose of review: this review highlights some of the recent concerning emerging infectious diseases, a number of them specifically that the world health organization has categorized as priorities for research. recent findings: emerging and reemerging infectious diseases account for significant losses in not only human life, but also financially. there are a number of contributing factors, most commonly surrounding human behavior, that lead to disease emergence. zoonoses are the most common type of infection, specifically from viral pathogens. the most recent emerging diseases in the usa are emergomyces canadensis, the heartland virus, and the bourbon virus. summary: in addition to the aforementioned pathogens, the severe acute respiratory syndrome, middle east respiratory syndrome, nipah virus, new delhi metallo-ß-lactamase-1 enterobacteriaceae, rift valley fever virus, and crimean-congo hemorrhagic fever virus are reviewed. these pathogens are very concerning with a high risk for potential epidemic, ultimately causing both significant mortality and financial costs. research should be focused on monitoring, prevention, and treatment of these diseases. in 1992, an expert committee that produced the institute of medicine report on emerging infections defined them as "new, reemerging, or drug-resistant infections whose incidence in humans has increased within the past two decades or whose incidence threatens to increase in the near future." additionally, six major contributors to these diseases were presented and included changes in human demographics and behavior, advances in technology and changes in industry practices, economic development and changes in land-use patterns, dramatic increases in volume and speed of international travel and commerce, microbial adaptation and change, and breakdown of public health capacity [1] . a common theme to recent emerging infectious diseases is that the majority are of animal origin [2] . in fact, some vector-borne pathogens that are projected to be introduced into new regions include rift valley fever and japanese encephalitis viruses in the americas and crimean-congo hemorrhagic fever virus in eurasia [3] . additionally, the majority of them have been viral. in addition to the cost of human life, emerging or reemerging infectious diseases can be very costly financially. the recent zika outbreak is estimated to have cost $3.5 billion in economic losses in 2016 [4] . the world health organization has prioritized a number of infectious diseases as requiring urgent need for research and development given the concern for potential of severe outbreaks. this article reviews the majority of emerging infections on this list, a few others that have emerged in the usa as well, and those that have not recently emerged but that the who has prioritized as having a high likelihood of causing outbreaks in the future. in 2002, an outbreak in southern china of atypical pneumonia of undetermined etiology spread to neighboring countries and eventually across the world. the coronavirus that caused the severe acute respiratory syndrome (sars) was then discovered [5] . hong kong and beijing were the most severely affected cities with estimated costs in the far east of this article is part of the topical collection on infectious disease * kevin watkins kwatkin4@gmail.com approximately $30 billion by may 2003 alone [6] . over 8000 probable cases were reported in 29 countries with a mortality rate of about 10% by the end of the epidemic in july 2003. sars reemerged in small scales from the end of 2003 to 2004. it is a positive-sense rna coronavirus whose mean incubation period is 5 days [7] . the clinical presentation varied by patient age: children developed typical mild upper respiratory tract infection while teenagers and adults developed a more severe but predictable course [5, 7] . phase i of the course was associated with increasing viral load with flu-like symptoms. phase ii was characterized by recurrence of fever with hypoxemia and progression of pneumonia. about 50% would require supplementary oxygen and approximately 20% would require intubation due to the development of acute respiratory distress syndrome (ards). watery diarrhea was a prominent extrapulmonary manifestation. additionally, hepatitis was a common complication and patients occasionally developed neurologic manifestations including status epilepticus. the clinical worsening in phase ii is thought to be immune-mediated. laboratory findings include lymphopenia, elevated aminotransferases, elevated lactate dehydrogenase (ldh), and creatine kinase (ck), with results consistent with disseminated intravascular coagulopathy. progression from unilateral opacities to bilateral involvement was found on imaging. sars is detected in respiratory secretions, urine, and feces [7] . however, the most notable finding is diffuse alveolar damage [5] . advanced age, severe hepatitis, high initial ldh, high neutrophilia on presentation, diabetes mellitus, low cd4 and cd8 counts, and high initial viral load are all prognostic factors for severe disease. some survivors have shown persistent lung function abnormalities. it is spread by close contact via droplet transmission, though there is some evidence that it might follow airborne transmission [7] . the virus has a high rate of infecting healthcare workers, accounting for approximately 20% of cases [8] . the original outbreak was thought to have started from the handling of wild animals, particularly the civet cat and raccoon dog [7, 9] . if suspected, a patient should be placed in respiratory isolation. a single test result, either positive or negative, is insufficient to make conclusions about a patient's diagnosis as both false positives and false negatives occur. it is initially tested for via serology or reverse-transcriptase-polymerase chain reaction (rt-pcr). however, two specimens obtained from either different sources or from the same source on different occasions confirm the diagnosis. specimens can be obtained from a nasopharyngeal swab or stool. additionally, seroconversion is another diagnostic method. it is vital to identify patients with sars, as isolation within 3 days of illness significantly reduces secondary transmission [10] . although ribavirin was used in the initial outbreak, it has not been shown to have in vitro activity against the virus. corticosteroids should be used only in the late-phase for rescue purposes as it may prolong viremia if given early [7] . most of the other agents that have shown promise, including monoclonal antibodies, nitric oxide, protease inhibitors, and interferons have not yet shown activity in humans and there have been very few animal studies [7, 11] . part of the concern about the potential for future epidemics lies in the fact that the family of viruses from which sars comes is notorious for frequent mutations [6] . additionally, there has been decreased worldwide attention, leading to decreased funding for research, as there have been no reported cases of sars since 2005 [11] . the middle east respiratory syndrome coronavirus (mers-cov) was first identified in 2012. it is a ß coronavirus that is enveloped with a positive-sense rna genome. the clinical features may include flu-like symptoms, gastrointestinal symptoms, severe pneumonia with acute respiratory distress syndrome, septic shock, disseminated intravascular coagulopathy, and multi-organ failure [12] . lab findings may include lymphopenia, thrombocytopenia, elevated ldh levels, and elevated aminotransferases [13, 14] . however, approximately 20% of cases had no or mild symptoms while almost 50% had severe symptoms [15••] . the incubation period is 2-14 days. children and younger adults seem to be less susceptible [14] . a large proportion of the confirmed pediatric cases have been asymptomatic and have been acquired via household contacts [16] . primary infection results from contact with a dromedary camel or its products, though human-to-human transmission is possible. cases acquired via human transmission tend to be more mild and the number of infected contacts tends to be limited. in fact, the majority of secondary cases have been associated with healthcare settings, where the majority of outbreaks have occurred and about 18% of cases have been healthcare workers [14] . with the exception of south korea, outbreaks have been limited to the middle east where dromedary camels are found [12] . in the south korean outbreak, there were 186 cases and 38 deaths [15••] . cases have been imported to europe, asia, africa, and the usa [ table 1 ]. the cases in the usa were detected promptly, so no secondary cases occurred. both cases occurred in may 2014 and were healthcare workers in saudi arabia [14] . as of july 2017, there have been 2040 laboratory-confirmed cases, with the majority of these in saudi arabia. the reproduction number is less than 1 with significant heterogeneity [15••, 17] . in the south korean outbreak, "spreaders" had significantly higher rates of underlying pulmonary disease. most transmissions occurred early in the disease course, during days 4-7 after symptom onset and were associated with patients going to multiple healthcare facilities before diagnosis [17] . human-to-human transmission seems to require close contact, though has not been sustained. most cases requiring hospitalization are those with chronic comorbidities, such as obesity, diabetes, end-stage renal disease, or chronic lung disease [13] . the mortality rate is about 35% [15••] . among those who were critically ill, patients frequently had underlying chronic disease, and extrapulmonary manifestations were common [18] . mers can be detected in respiratory tract secretions with bronchoalveolar lavage specimens providing a better yield than nasopharyngeal swabs; however, serum samples may also be obtained [13] . the centers for disease control and prevention (cdc) recommends evaluation for patients with respiratory symptoms and travel to an affected country within 14 days or for those who are in a cluster of patients with severe acute respiratory disease in which mers-cov is being evaluated [14] . testing should not be performed before 24 h of exposure [19] . healthcare workers with exposure during aerosol-generating procedures are at high risk for transmission; because of this, the cdc recommends airborne and contact precautions for suspected or known cases [14] . patients suspected to have mers should be admitted if they have shortness of breath, pneumonia, or hypoxemia. otherwise, they may be cared for at home in isolation; however, these cases should be carefully selected for potential of spread [19] . treatment is primarily supportive with mechanical ventilation and renal replacement therapy as needed as there are no specific antivirals that have been developed [13] . a number of studies have been performed with a variety of medications, including interferons (ifn), ribavirin, protease inhibitors, mycophenolic acid, cyclosporin a, chloroquine, nitazoxanide, antibiotics, fusion inhibitors, and steroids. perhaps the most promising human studies have involved a combination of ribavirin and interferons, though overall results have been mixed. some have shown no benefit, but have been critiqued that treatment was too late. other studies have shown improved 14-day survival but not at 28 days, and others have shown improved survival at both [12, 20, 21] . a small retrospective study had all eight patients who received combination therapy with ifn-ß and mycophenolic acid survive, though they had lower acute physiology and chronic health evaluation ii scores compared to patients who received other agents [21, 22] . two new viruses that have recently been discovered in the usa are the heartland and bourbon viruses. the heartland virus is a member of the bunyaviridae family in the genus phlebovirus with single-stranded, negative-sense rna [23] . the lone star tick (amblyomma americanum) transmits the disease [24] . it was first discovered in 2009 from a farmer in missouri. clinical features include flu-like symptoms, leukopenia, thrombocytopenia, and mild transaminitis and develop within 14 days of tick bite. six additional cases were identified during 2012-2013; four of these patients were hospitalized and one patient with multiple underlying diseases died. five patients were in missouri and one was in tennessee [23, 24] . the deceased patient had developed hypoxemic respiratory failure with severe thrombocytopenia, acute kidney injury, and upper gastrointestinal bleeding and his family ultimately opted to transition to comfort measures. his clinical course was consistent with severe infection from ehrlichia chaffeensis except that he did not improve with doxycycline [25] . diagnosis should be suspected when a patient does not improve with doxycycline therapy and can be made with detection of viral rna by rt-pcr [26•]. the bourbon virus is in the genus thogotovirus within the family orthomyxoviridae and is pleomorphic with an rna genome. the case patient was from bourbon county, kansas, in 2014. within several days of tick bite, he developed flu-like symptoms. later findings included leukopenia, thrombocytopenia, hyponatremia, and mild transaminitis; the case patient then developed congestive heart failure and later died after withdrawal of care [27] . a study of tick populations in missouri revealed that amblyomma americanum ticks were carriers of the virus, though transmission from these ticks is not certain [28] . little is known otherwise about the disease as confirmed cases have been scarce. nipah encephalitis was first noted in an outbreak of fatal encephalitis starting in 1998 in malaysia. it is an rna virus from the family paramyxoviridae, genus henipavirus. the outbreak initially involved pig-farming villages but spread to other areas [29] . since discovery, outbreaks have been recognized almost yearly in bangladesh and occasionally in india [30] . it is characterized by a high attack rate with neurologic features at presentation. the strain in malaysia rarely displays respiratory symptoms, though the more concerning bangladeshi strain often has a respiratory component [29, 30] . prodromal symptoms consisting of sore throat, myalgias, fever, headache, and vomiting are common with the ultimate development of altered mental status and potentially seizures and coma. the majority of patients show brainstem dysfunction with pinpoint pupils, dysautonomia, and abnormal vestibulo-ocular reflex. myoclonus may also be present and characteristically involves the diaphragm and anterior neck muscles. patients commonly develop thrombocytopenia and abnormal liver function tests. cerebral spinal fluid analysis yields normal glucose with elevated white cell counts and protein typical of viral infections. diagnosis can be made by rt-pcr. most patients with encephalitis had abnormal electroencephalograms and magnetic resonance imaging studies (mri). mris commonly show small discrete hyperintense lesions in the subcortical and deep white matter [29] . pathophysiologically, nipah virus causes a widespread vasculitis most commonly involving the brain and lungs [30] . mortality rates have been quoted to be between 39 and 70%. the majority of people infected develop severe disease. neuropsychiatric sequelae are not uncommon (one third of survivors) with persistent cognitive impairment, cerebellar signs, and peripheral nerve lesions [29, 30] . relapse and late-onset encephalitis may also occur months or years after the acute illness, though rates are rare (about 5%). additionally, relapse or late-onset disease has a lower mortality rate of about 18% with minimal brainstem involvement. transmission can consist of consumption of food contaminated by bat saliva (raw sap is common), contact with infected animals, or human-to-human spread [29, 30] . sustained human-to-human transmission beyond 5 generations has not been recognized and the reproductive number has averaged 0.48. those that are at greatest risk of infection are providers of fatally infected patients with prominent respiratory secretions. however, most patients do not transmit infection to anyone [30] . bats of the family pteropodidae are the natural reservoirs, though numerous mammals can become infected. treatment consists of supportive care. treatment with ribavirin was tried during a malaysian outbreak with 36% reduction in mortality; however, this was not a controlled clinical trial and did not reach statistical significance [29] . the concern about a potential for pandemic arises from the fact that it is an rna virus and the bangladeshi strains have high genetic variability with potential for an increase in the reproductive number [30] . an experimental human monoclonal antibody (m102.4) has shown promise in nonhuman studies [31] . another emerging infectious disease is the dimorphic fungus, emergomyces canadensis. although other emergomyces species have been discovered throughout europe and africa, e. canadensis was only recently discovered in north america [32] . emergomyces was previously named "emmonsia-like" pathogens. there are numerous species of emergomyces, with the most common being e. africanus that causes infections in those with hiv in south africa. no animal infections have been documented [33] . there have been four cases noted so far in immunocompromised patients that developed systemic disease with fungemia. patients commonly had pneumonia as well as skin lesions. it is suspected that inhalational infection occurs and that geographic range involves central and western north america [32] . the diagnosis is made via histopathology or culture of affected tissue [33] . limited susceptibility testing has shown susceptibility to itraconazole and amphotericin b. treatment should follow typical guidelines for dimorphic fungal infections in immunocompromised hosts with amphotericin b for 1-2 weeks followed by itraconazole for 12 months [32] . one of the most concerning emerging infectious diseases is the n e w d e l h i m e t a l l o -ß -l a c t a m a s e -1 ( n d m -1 ) enterobacteriaceae. these "superbugs" seemed to have originated in the indian subcontinent and cases were first reported in 2008, though retrospective analysis shows its presence as early as 2006. the ndm-1 gene is carried on a plasmid, bla ndm-1 , that is easily spread among bacteria [34] . although carbapenamases are not new, the level of promiscuity of ndm-1 is higher than others [35] . additionally, studies have shown both a high level of inter-lineage and inter-species gene transfer though the dominant mechanism seems to be the latter. it is most commonly found among escherichia coli and klebsiella pneumoniae, but has also been found within all species of enterobacteriaceae as well as acinetobacter baumanii and pseudomonas species [34] . although gram-negative bacteria have typically been treated with ß-lactams, fluoroquinolones, and aminoglycosides, ndm-positive pathogens contain up to 14 antibiotic resistance genes and are only susceptible to tigecycline and colistin, with the exception of a few strains sensitive to aztreonam and certain aminoglycosides [36] . multiple variants have been reported (i.e., ndm-2). ndm-1 is widely found throughout both hospital strains as well as community strains in india, which is consistent with the rates of extendedspectrum ß-lactamase (esbl) of 79% in both the community and hospital setting [34, 35] . it is likely that the overuse of antibiotics led to excretion into the waste water systems, ultimately resulting in selection-pressure for multidrug-resistant organisms [36, 37] . in fact, some estimate that at least 100 million indian residents have gut flora consisting of ndm-1containing bacteria [37] . ndm-1 has spread to numerous other countries; it is believed that the medical tourism industry propagated its dissemination. in fact, seven out of eight tourists to india were colonized with esbl-positive bacteria upon return, which they did not have upon departure [35] . the balkans have been another area with high rates of ndm-positive bacteria, which has now spread to all continents except south america and antarctica [ fig. 1 from reference 34] ]. ndm-positive bacteria have even now been found in companion animals in the usa [34] . adding to the concern is that there are few antimicrobials in the research pipeline with activity against gram-negative pathogens. rift valley fever virus (rvfv) is a single-stranded rna virus of the genus phlebovirus, family bunyaviridae. the majority of infected individuals are asymptomatic. most patients who are symptomatic have a self-limiting febrile illness; however, a small minority develop severe disease with acute hepatitis, renal failure, and hemorrhagic manifestations. those who survive this phase often develop neurologic symptoms consisting of vision loss and encephalitis. long-term sequelae may develop with blindness, hemiparesis, quadriparesis, incontinence, hallucinations, or coma. the fatality rate is 1-3%, though patients with hemorrhagic disease have a fatality rate as high as 50%. diagnosis is made by detection of viral rna and immunoglobulin serology, particularly during the acute febrile stage [38] . it is often transmitted by mosquitoes, typically of the aedes genus, though exposure to infected animal tissue or consumption of their products can also cause disease [39] . rvfv is endemic in sub-saharan africa with periodic outbreaks after heavy rainfall and flooding. it has spread to the middle east and the french island of mayotte. outbreaks often have significant effects on both humans and livestock. characteristics of an rvfv outbreak include the sudden increase in abortions and mortality of young animals with the appearance of disease in humans. models have been developed with successful predictions of outbreaks using satellite imagery of sea surface temperatures, rainfall, and vegetation. given this spread out of africa, there is concern about it reaching the mediterranean basin and europe. a number of competent vectors for rvfv have been identified in europe. it has additionally been isolated from other hematophagous arthropods including ticks and sand flies, though their significance is not currently understood. there is currently no specific treatment, though a vaccine, tsi-gsd-200, has provided protection following primary inoculation and single boost schedule [38] . crimean-congo hemorrhagic fever virus (cchfv) produced its first major known outbreak from 1944 to 1945 in the crimean peninsula, though it has likely been causing outbreaks for centuries. it was not identified until 1968. as such, it has not recently emerged but is listed as a priority agent for research and development by who due to its potential for spreading and causing more severe outbreaks. cchfv is a member of the bunyaviridae family, genus nairovirus. it has many aliases, including asian ebola, karakhalak (black death), khunymuny (nose bleeding), and khungribta (blood taking). cchfv derives its name from outbreaks both in crimea and the belgian congo and has been reported throughout many parts of africa, the middle east, europe, and asia [ fig. 2 from reference 40 ]. crimean-congo hemorrhagic fever (cchf) is the most widespread tick-borne viral infection of humans [41] . it is a negative-sense rna virus transmitted by numerous tick species; because of this, agricultural workers are most commonly affected [40, 41] . additionally, human-to-human transmission may occur via contact with skin, mucous membranes, or body fluids of infected patients [40] . standard barrier precautions are recommended for patients with suspected disease [41] . domestic livestock are the main reservoirs. outbreaks have been increasing in recent years. likely targets of the virus are endothelial cells, hepatic cells, and kupffer cells. the clinical course of cchf follows 4 phases consisting of incubation, prehemorrhagic, hemorrhagic, and convalescence [40] . there is a spectrum of severity from a mild, febrile illness to hemorrhagic shock with multiorgan failure [41] . the incubation period varies by method of acquisition; it typically takes 1-3 days by tick bite and 5-6 days by exposure to infected patients. the prehemorrhagic stage is nonspecific with flulike symptoms. the hemorrhagic stage follows and develops within 3-6 days of symptoms in severe disease [40] . one differentiating feature is that severely ill patients often develop a pattern of large ecchymosis not seen in other hemorrhagic fevers [41] . laboratory findings include leukopenia, thrombocytopenia, elevated aminotransferases, elevated ck and ldh, fig. 2 distribution of crimean-congo hemorrhagic fever virus and prolonged prothrombin time and activated partial thromboplastin time. convalescence usually occurs 15-20 days after onset of illness [40] . diagnosis is commonly made via detection of viral rna with rt-pcr or serology. there has traditionally been no specific treatment for cchf, though ribavirin has demonstrated both in vitro and in vivo activity [40] . furthermore, both the cdc and who recommend use of ribavirin [42•] . a report from turkey claimed that a combination of methylprednisolone, intravenous immunoglobulin (ivig), and freshfrozen plasma was beneficial, but there was no control group for comparison [41] . ivig and methylprednisolone may be beneficial by improving thrombocytopenia [42•, 43] . two vaccines have been developed, though they have not been through randomized clinical trials [40] . cchf has never been demonstrated in vaccinated individuals and the bulgarian ministry of health has reported a fourfold decrease in cases since vaccine implementation [41, 43] . ribavirin might be beneficial for post-exposure prophylaxis [43] . mortality rates have varied between 10 and 50%, with worsening prognosis if the disease is acquired nosocomially, with higher levels of aminotransferases, higher viremia, or with coagulopathy [40, 41] . long-term sequelae have been documented but are rarely permanent and include impaired vision and other neurologic symptoms [42•] . cchf has epidemic potential with high rates of mortality, nosocomial infection, and difficulty with prevention and treatment [40] . additionally, it is the most genetically diverse of the arboviruses [41] . attempts to control cchf via eradication of tick vectors have proven inefficient and domestic animals are asymptomatic even when highly viremic [42• ]. infectious diseases promise to be one of the biggest challenges in the coming decades, with new ones emerging unpredictably. successful clinical outcomes require a proactive approach with research and development, as human behavior contributes to new infections emerging, particularly with zoonoses. although this strategy would prove costly, this article has demonstrated a few examples of how much an emerging infectious disease can cost during an outbreak. this article has reviewed a number of emerging infections that the who has categorized as priorities for research and development due to their potential for epidemics or even a pandemic. additionally, it has reviewed a concerning drug-resistance mechanism that threatens to provide bacteria with resistance to all antibiotics. finally, a few infectious diseases that have emerged in the usa have been covered, though little is known about them, with a paucity of cases to date. conflict of interest the author declares that he has no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. emerging infectious diseases: a cdc perspective emerging infectious diseases in 2012: 20 years after the institute of medicine report emerging infectious disease: a proactive approach a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome (sars): a review of the history, epidemiology, prevention, and concerns for the future severe acute respiratory syndrome and coronavirus severe acute respiratory syndrome (sars) severe acute respiratory syndrome and influenza: virus incursions from southern china who guidelines for the global surveillance of severe acute respiratory syndrome (sars) anti-sars coronavirus agents: a patent review (2008-present) middle east respiratory syndrome coronavirus: a comprehensive review middle east respiratory syndrome: what clinicians need to know middle east respiratory syndrome coronavirus: update for clinicians this article gives the majority of a summary of perhaps most significant recent emerging infectious disease in terms of both mortality and number of cases middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus a review of treatment modalities for middle east respiratory syndrome treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia a new phlebovirus associated with severe febrile illness in missouri notes from the field: heartland virus disease-united states heartland virus associated death in tennessee update and commentary on four emerging tick-borne infections novel thogotovirus associated with febrile illness and death bourbon virus in field-collected ticks nipah encephalitis-an update the pandemic potential of nipah virus therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody emergomyces canadensis, a dimorphic fungus causing fatal systemic human disease in north america emergomyces: a new genus of dimorphic fungal pathogens causing disseminated disease among immunocompromised persons globally global spread of antibiotic resistance: the example of new delhi metallo-ß-lactamase (ndm)-mediated carbapenem resistance the new medical challenge: why ndm-1? why indian? emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study the emergence of pan-resistant gramnegative pathogens merits a rapid global political response rift valley fever virus: a review of diagnosis and vaccination, and implications for emergence in europe risk factors associated with human rift valley fever virus infection: systematic review and meta-analysis crimean-congo hemorrhagic fever crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity crimean-congo haemorrhagic fever virus: past, present and future insights for animal modelling and medical countermeasures recent advances in research on crimean-congo hemorrhagic fever key: cord-022046-q1exf47s authors: toosy, arshad haroon; o'sullivan, sean title: an overview of middle east respiratory syndrome in the middle east date: 2018-09-28 journal: fowler's zoo and wild animal medicine current therapy, volume 9 doi: 10.1016/b978-0-323-55228-8.00042-4 sha: doc_id: 22046 cord_uid: q1exf47s nan middle east respiratory syndrome (mers) is an emerging infectious zoonotic disease caused by a novel coronavirus (cov). mers was first reported in 2012 in jeddah, kingdom of saudi arabia (ksa), and in jordan, respectively. 1 the disease was considered a potential pandemic threat to public health in the persian gulf region. 2 (see also chapter 19.) most known covs infect and circulate in animals, mainly bats, but a number of covs are known to cause human disease (see also chapter 40). [3] [4] [5] the rapid emergence of mers-cov coupled with its limited geographic distribution has led to the suspicion that this is a zoonotic disease with an animal reservoir, 4 and the evidence supports the hypothesis that dromedary camels (dcs) are the reservoir host. in dcs mers-cov causes a mild, transient upper respiratory tract (urt) infection. [4] [5] [6] a mild or asymptomatic disease has also been reported in humans, but this is not always the case. mers-cov infection in humans often results in a severe, life-threatening disease of the lower respiratory tract (lrt), with high mortality. 1, 2, 5 immunocompromised, elderly people and those with comorbidities, usually with a history of close contact with infected dcs, are particularly susceptible. 7 is a positive-sense enveloped single-stranded rna virus and is the first lineage of 2c betacoronavirus known to infect humans. 2, 8 it is more closely related to bat covs hku4 and hku5 (lineage 2c) than to the severe acute respiratory syndrome cov (sars-cov, (lineage 2b). 2,3 recent genome sequencing analysis reported the genomic evolution rate (1.12 × 10 3 substitutions per site), suggesting that mers-cov diverged from its viral ancestor in march 2012. 12 analysis of human mers-cov sequences has identified several circulating genotypes. these distinct genotypes are phylogenetically classified into clades a, b, and, most recently, c, which correlate with outbreaks of mers among humans. 4, 5, 8, 12 the emergence of divergent mers-cov clades in humans since 2012 is consistent with several independent sporadic introductions into the human population from an animal reservoir, of which the camel was unquestionably the source. 6, 8, 12, 13 pathogenesis host cell entry of mers-cov is mediated by the binding of mers spike (s) proteins to a specific cellular receptor known as dipeptidyl peptidase 4 (dpp4). 11 dpp4 is expressed on the epithelial and endothelial cells of most human organ tissues in ex vivo studies using human tissue culture lines; this may account for the multisystem clinical spectrum of the mers-cov infection. 2, 14 a strain cultured from a fatal human case was experimentally inoculated into three dcs using intratracheal, intranasal, and conjunctival routes. 15 a mild transient disease resulted in submucosal inflammation and necrosis in the urt and lrt, but the alveoli remained unaffected. 15 experimental inoculation of rhesus macaques (macaca mulatta) and common marmosets (callithrix jacchus) resulted in mild to severe lrt disease causing multifocal interstitial pneumonia in the macaques and extensive fatal pneumonia in the marmosets. 2, 14, 16 since at least 1992. 13 several mers-cov serologic surveys confirm that the disease is not present in domesticated livestock (namely, horses, sheep, goats, and cattle) and is enzootic in the dc population across the arabian peninsula as well as in north and east africa. 6, 7, 13, 15, [19] [20] [21] [22] [23] historically, the camel was the mainstay of land-based trade transportation and was used extensively as a food source across the entire region prior to industrialization during the latter part of the 20th century. 5, 10 the free movement of humans and animals across the region supports the widespread prevalence and genetic diversity of mers-cov in the dc populations of arabia and east africa today. 5, 10 the temporal dynamics of mers infection in dcs in al-ahsa, ksa, was examined by collecting nasal swabs and lung tissue during postmortem examination from two independent groups of animals over the course of a year and testing these for mers-cov rna by real-time reverse-transcriptase polymerase chain reaction (rt-rtpcr). 24 positive samples were typically associated with young immunologically naive animals (<4 years of age) rather than adults (>4 years of age). seasonal peaks were detected during the winter months and coincided with the calving season, less extreme environmental conditions, cooler ambient temperatures, and higher relative humidity, for the transmission of infection amongst susceptible individuals. 24 this seasonal peak has also been described in epidemic nosocomial outbreaks in humans that occur more frequently during the winter months. 25, 26 extensive virologic evidence has been accumulated since 2012 supporting the epidemiologic link between dcs and humans in the transmission of mers-cov, although epidemiology mers-cov belongs to a lineage commonly associated with bats, the closest relatives of which lineage were recently identified in vesper bats (i.e., various species of the family vespertilionidae) from europe, asia, and south africa. initial research efforts have focused on establishing an epidemiologic link between bats and humans. 3, 4, 6, 17 there is no conclusive evidence to support the theory that bats are the source of human infection, although there is consensus that bats are the ancestral hosts of the disease. 4,5,17 a related mers-like cov virus, isolated from an african pipistrelle bat (pipistrellus hesperidus) in uganda, has shown high divergence of the s protein nucleotide sequence compared with an index mers-cov s protein sequence (46% amino acid identity divergence). 17 this suggests that the two viruses differ significantly in receptor binding properties, implying that the mers-like cov virus is not a zoonotic threat and supporting the theory of a common ancestry. 17 to date the only direct link between bats and human disease was a single instance when an rna sequence from a fatal human mers index case showed a 100% nucleotide match of a polymerase chain reaction (pcr)-amplified sequence of a fecal pellet from an egyptian tomb bat (taphozous perforates) collected in the same area of bisha, ksa. 18 the human fatality was an owner of four dcs, which also tested positive for the same strain of mers-cov. 18 at present, bat-to-human infection by mers-cov is considered to be purely speculative. 4 surveillance of dcs in ksa has shown that mers-cov clade b has been enzootic in the camel population in arabia genetic deep sequencing methods (i.e., high-throughput sequencing) have been readily available to researchers since the disease was first reported. 12 sequenced data have been used in these cases to successfully construct the phylogenetic tree between related viruses and hosts. 2, 6, 7, 12 direct mers-cov antigen detection is possible but has been rarely performed. 10 immunochromatography assays and monoclonal antibody-based capture elisas targeting the mers-cov nucleocapsid protein have been described. 20 since the virus was first reported in 2012, a range of comprehensive laboratory tests has been developed. 10, 30 to better understand the disease, it has been important to collate sampling methodology data, laboratory results, and analyses in combination with clinical and epidemiologic data. 10 until laboratory assays are fully validated, a combination of molecular and serologic laboratory tests is required to improve confidence in laboratory diagnosis during outbreaks. 30 in cases of mild or asymptomatic infection, full validation of serologic assays is required to rule out false-negative results. 31 validation is also required to successfully apply newly developed diagnostic serology algorithms to inform public health decisions. 10, 30, 31 treatment therapeutic options for mers-cov are limited. supportive treatment is indicated for hospitalized patients, but vigilance for complications is essential. 8 empirical use of antimicrobial agents or steroids has not succeeded in reversing the progression of severe disease. 8, 27, 29 no specific drug or vaccine is currently available to treat mers. indeed, it has been stated that the complexity and time required for the development and registration process of drugs for human use impedes the ability to counter the rapid threat against an emerging infectious agent. 8 for example, there is no vaccine available against sars-cov because of the brevity of the threat to the public health. 2, 8 it is likely that a mers-cov vaccine for human use may not be developed due to a lack of commercial interest, or if the threat posed by mers-cov declines in the meantime. 8 nevertheless, given the prevalence of mers-cov infection in the middle east's dc population and due to the potential for spillover to the human population in direct contact with dcs, the development of a vaccine for use in dcs may be feasible. 4, 5, 32 a recent successful trial of a mers orthopoxvirus vaccine has conferred mucosal immunity in the urts of dcs. 32 eradication of mers-cov from herds may be possible, if vaccines are administered to young, immunologically naive camels prior to exposure. 4, 5, 32 identification of the zoonotic source of mers guides control strategies at the human-animal interface. 3, 30 by preventing spillover of mers-cov from animals to humans, the risk of nosocomial and familial outbreaks in the middle east could be eliminated. 3 strategic serosurveys of humans using samples collected after 2012 have been infrequent. 4, 5, 10 there is a paucity of baseline data to describe the proportion of the potentially infected human population for much of the arabian peninsula and all of east africa, including the horn of africa. 10 the exact mechanism of transmission from camels to human remains uncertain. 10 sustained close contact is most probably necessary for transmission by aerosolized droplets, as mers-cov viral rna has been detected in air samples from a barn housing infected dcs in qatar, and the virus may remain viable in aerosol for up to 45 minutes. 10, [27] [28] [29] the potential public health risk resulting from aerosol-generating activities ranges from contamination of a room occupied by a symptomatic patient to slaughter practices. 8, 24, 30 aerosolized transmission of mers-cov has been attributed to hospital outbreaks in ksa and south korea. 26, 27, 29 mers-cov spreads inefficiently from human to human, but transmission is effective in a hospital environment, where susceptible individuals are concentrated and the risks are amplified by poor infection prevention and control (ipc) protocols. 8 in some reported cases of mers, direct contact with camels was not apparent. 27, 29 camel-to-human transmission through other routes is, however, possible owing to the consumption of unpasteurized camel milk or raw camel meat and in traditional medicine, when camel urine is consumed as a natural remedy for a variety of ailments. 3, 10 a recent survey has found that infected camels may shed mers-cov virus in milk and urine, and the virus has been shown to remain infectious for 3 days in milk stored at 4°c. 3, 10 the transmission risks associated with the handling of camel products, raw milk, urine, and meat during animal slaughter are yet to be fully elucidated. further studies are needed to demonstrate the potential of camel-to-human transmission. 8 serologic methods with high sensitivity and specificity to detect mers-cov antibodies have been developed for use in seroepidemiologic studies. methods include indirect immunofluorescence assays, enzyme-linked immunosorbent assays (elisas), protein microarray technology, and microneutralization (mn) assays. 13, [20] [21] [22] pseudoparticle virus neutralization tests (ppnts) and conventional mn assays have also been used to detect neutralizing antibodies to mers-cov. 18 validated molecular assays have been developed. 10, 13, 19 rt-rtpcr is the preferred diagnostic method for the detection of mers-cov and has been endorsed by the who. 1 confirmation of mers in suspected cases requires the screening of samples targeting a number of genes specific to mers-cov, namely up e, orf 1a, orf 1b, and n genes. 1, 10, 13, 19 its infancy. aside from bats, the role that other wildlife may play in the ecology of mers-cov in east africa and arabia is yet to be elucidated. at present the implementation of intensive ipc measures in human health care is vital, including improving education and awareness among healthcare workers. 1, 8 most human cases have been linked to lapses in ipc, as one-fifth of viral infections have been reported among healthcare workers. 1, 8 stringent precautions while handling suspected mers-cov patients include the use of personal protective equipment (ppe) (i.e., disposable gloves, gowns, respiratory protection, and eye protection). 2, 8, 33 immunocompromised individuals and those with preexisting medical conditions should avoid close contact with dcs. 2, 8 public health authorities should adopt a standardized public health response protocol to include standardized case reporting methodology as defined by the who. 1, 30 standardization of case definitions aids accurate calculation of a case fatality ratio by including mild or asymptomatic cases. 30 the health authority of abu dhabi in the united arab emirates recently implemented a standardized reporting option for mers, successfully incorporating it into existing epidemiologic surveillance systems with the aim of enhancing surveillance, educating healthcare workers, and ensuring laboratory capacity. 25 in countries where mers-cov is enzootic in dcs, mers control at the animal-human interface is unlikely to succeed unless appropriate preventive strategies are implemented. 5 these should include the following: • strict regulation of camel movement with imposition of a requirement for mers clearance prior to the importation and transport of camels, including animals presented for slaughter. • camels with detectable mers-cov rna should be quarantined and tested at regular intervals. • use of appropriate ppes while handling dcs. • increased awareness among camel owners and the general public of the risks of consuming unpasteurized camel milk and urine. this may prove challenging given the depth of customs and beliefs in some areas. • accelerated development of safe and effective mers vaccines for animal and human use. 5, 33 conclusions mers-cov has been observed for only 4 years, and vigilance is vital for the containment of the disease due to the high case fatality rate in humans and possible genetic instability of the virus. 8 continued laboratory testing, genetic sequencing, analysis, timely data sharing, and clear communication are essential if such vigilance is to be effective. 8 nonetheless, despite the potential for a pandemic outbreak at multiple mass gatherings during the islamic calendar (hajj, eid, and umrah) there were no reported outbreaks of mers during or immediately after these events. 10 as such mers-cov is not a virus of pandemic concern. 10 since 2012 our understanding of mers has increased greatly although gaps in knowledge still exist. the understanding of the disease's ecology-especially the interplay between camels, humans, and the environment-is still in who mers-cov global summary and risk assessment middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd who emergencies: mers-cov. available at mers coronavirus: diagnostics, epidemiology and transmission dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia pathogenesis of middle east respiratory syndrome coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels response to emergence of middle east respiratory syndrome coronavirus transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital outbreak of middle east respiratory syndrome coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient mers-cov outbreak in jeddah-a link to health care facilities mers coronavirus: data gaps for laboratory preparedness asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels centers for disease control and prevention: interim prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). available at an animal model of mers produced by infection of rhesus macaques with mers coronavirus further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia antibodies against mers coronavirus in dromedaries middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia the authors wish to thank the following: h. e. ghanim mubarak al hajeri and the senior management of al ain zoo for their encouragement and support, dr. ahsan ul haq of dubai camel hospital for his technical insight, and dr. andrew higgins, fellow of the zoological society of london and honorary editor in chief of the veterinary journal, who reviewed the manuscript. key: cord-318872-0e5zjaz1 authors: park, ji-eun; jung, soyoung; kim, aeran; park, ji-eun title: mers transmission and risk factors: a systematic review date: 2018-05-02 journal: bmc public health doi: 10.1186/s12889-018-5484-8 sha: doc_id: 318872 cord_uid: 0e5zjaz1 background: since middle east respiratory syndrome (mers) infection was first reported in 2012, many studies have analysed its transmissibility and severity. however, the methodology and results of these studies have varied, and there has been no systematic review of mers. this study reviews the characteristics and associated risk factors of mers. method: we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) for englishor korean-language articles using the terms “mers” and “middle east respiratory syndrome”. only human studies with > 20 participants were analysed to exclude studies with low representation. epidemiologic studies with information on transmissibility and severity of mers as well as studies containing mers risk factors were included. result: a total of 59 studies were included. most studies from saudi arabia reported higher mortality (22–69.2%) than those from south korea (20.4%). while the r(0) value in saudi arabia was < 1 in all but one study, in south korea, the r(0) value was 2.5–8.09 in the early stage and decreased to < 1 in the later stage. the incubation period was 4.5–5.2 days in saudi arabia and 6–7.8 days in south korea. duration from onset was 4–10 days to confirmation, 2.9–5.3 days to hospitalization, 11–17 days to death, and 14–20 days to discharge. older age and concomitant disease were the most common factors related to mers infection, severity, and mortality. conclusion: the transmissibility and severity of mers differed by outbreak region and patient characteristics. further studies assessing the risk of mers should consider these factors. middle east respiratory syndrome (mers) was first reported in 2012 in saudi arabia [1] . although most patients are linked to the arabian peninsula geographically, mers has been detected in many other parts of the world [2] . a large mers cluster was also observed in 2015 in south korea [3] . mers causes sporadic infection and intrafamilial and healthcare-associated infection. its symptoms can vary from asymptomatic infection to death. despite the infection's association with high mortality, specified antiviral therapy is lacking, especially for patients with concomitant diseases [2] . many previous studies have assessed the risks of mers, such as factors dictating severity or an infection risk, yet the indices they present vary. for example, the case fatality rate was found to be 25.9% in the middle east area, but 20.4% in south korea [4] . the incubation period was reported to be 6.83-7 days in south korea [4, 5] , but 5.5 in a study using data from multiple areas [6] and 5.2 in saudi arabia [7] . accurate assessment of the risk of mers is essential for predicting and preventing infection. a systematic review of the risk of mers, as covered in previous studies, is potentially helpful for predicting this spread, and its future impact. this study aimed at reviewing the risk of mers, focusing on indices related to infectivity and severity. we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) using the term "mers" or "middle east respiratory syndrome", encompassing articles published after 2000. the search process was conducted in october 2017. we also manually searched the reference lists of the included studies. human studies were included, while animal studies and reviews were excluded. only articles in english or korean were included. even if a study collected data on humans, such as collecting specimens from religious pilgrims, it was excluded if there were no mers patients in the study sample. additionally, case studies including fewer than 20 mers patients were excluded as they were considered as having insufficient mers patient numbers and representative information. the included studies were classified as epidemiologic studies and those covering risk factors of mers. in the epidemiologic category, indices related to the risk of mers were divided into two categories; related to infectivity and related to severity. the index related to infectivity included the reproduction number (r), attack rate, incubation period, serial interval, and days from onset to confirmation. the index related to severity included the case fatality rate (cfr), days from onset to hospitalization, days from onset to discharge, days from onset to death, and days from hospitalization to death. in the risk factor category, factors related to infection, transmission, severity, and mortality of mers were analysed. even if the included studies investigated factors that were related to mortality, when they did not analyse risk factors of severity or mortality using appropriate statistical methods (e.g., regression analysis, cox proportional hazards model) or only compared prevalence factors, we excluded them from the risk factor category. in all categories, we extracted the study period, number of participants, and geographical region where the data were collected using a data extraction form confirmed after pilot assessment. a total of 3009 studies were searched, and 2717 were reviewed, excluding 292 duplicate studies. after the title and abstract review, a further 1804 and 663 were excluded, respectively. another four studies were included via a manual search, which left a total of 58 studies for analysis ( fig. 1 ). the 38 of total 58 included studies were classified as epidemiologic studies (table 1) . r value, representing the reproduction number, indicates the average number of secondary cases generated by infectious individuals. thirteen studies reported r value of mers. four studies that used data from multiple areas had r < 1.0 [6, [8] [9] [10] . studies using saudi arabia or middle east area data reported r < 1, at 0.45-0.98 [11] [12] [13] [14] , though one reported 1.9-3.9 [15] . studies using south korea data showed higher values, at 2.5-8.09 [16] [17] [18] [19] , in the early stage, and < 1 in the later period [20] or with control intervention [19] . a total of eight studies reported the attack rate. four reported the overall or secondary attack rate, and the other four reported the attack rate of specific participant groups. two studies conducted in saudi arabia showed 0.42% [21] and 4% [22] secondary attack rates. studies in south korea showed secondary attack rates of 3.7% in one study [23] and 14.3-15.8% in another [24] . two studies reported the attack rate among healthcare workers (hcws). one study in south korea reported a mers incidence of 1.5% among hcws [20] , and another study using multiple area data reported a 13.4-13.5% infection rate among hcws [8] . the attack rate among hospital patients was 4% in one study [5] and 22% in the early and 1% in the later period in another [16] . the incubation period is the period between infection and appearance of signs of a disease. a total of 12 studies reported the incubation period of mers. nine used data from south korea and showed a 6-7.8 day incubation period [3] [4] [5] [23] [24] [25] [26] [27] [28] . one study using data from saudi arabia reported a 5.2 day incubation period [7] , and another using data from multiple areas reported a 5.5 day incubation period [6] . sha et al. compared the incubation periods between the middle east area and south korea and reported 4.5-5 and 6 days, respectively [29] . the serial interval of an infectious disease represents the duration between symptom onset of a primary case and of its secondary cases. two studies used south korea data, reporting serial intervals of mers of 12.6 and 14. 6 days, respectively [24, 26] . among five studies reporting days from onset to confirmation, three studies used data from south korea. one study analysing all south korea cases reported 5 days from onset to confirmation [3] . park et al. reported 6.5 days for all cases, 9 for second generation and 4 for third generation [28] . one study from taiwan reported 6 days for hcws and 10 for non-hcws [30] . a study from saudi arabia reported 4 days from onset to confirmation [31] . sha et al. compared the data from middle east and south korea areas and reported 4-8 and 4-5 days, respectively [29] . two studies from saudi arabia reported days from onset to hospitalization. one reported 2.9-5 days [32] , and the other reported 5.3 days [33] . twenty-six studies reported on mers-related mortality. ten reported the mortality rate in south korea as 14.5-47.8% [3, 4, 23-26, 28, 29, 34, 35] ; one of which, including all mers patients in south korea, reported a mortality rate of 20.4% [27] . ten studies analysing data from saudi arabia reported higher mortality rates, of 22-69. 2% [7, 12, 22, [31] [32] [33] [36] [37] [38] [39] , although others reported mortality rates 10% [40] and 19.9% [21] . a taiwanese study reported a mortality rate of 35.6% [30] . studies using data from multiple areas reported mortality rates ranging from 26.6% [29] to 59.4% [9, 41] . three studies reported days from mers onset to discharge. sha et al. reported 14 days in the middle east area and 17 in south korea [29] . one study from saudi arabia reported 17 days [36] , and another in south korea reported 20 [3] . two korean studies reported similar periods of 11-13 days from onset to death: 11-12.5 in park et al. [24] and 13 in ki et al. [3] . although one study from saudi arabia reported longer than 17 days from onset to death [36] , sha et al., comparing data between the middle east and south korea, reported similar periods of 11.5 and 11 days, respectively [29] . one taiwanese study also reported a similar period of 12-13 days [30] . two studies reported a similar length of hospitalization: 15 [33] and 15.2 days [19] . of the 20 studies included in the risk factor category, four were duplicates of studies in the epidemiologic category as they had information regarding the epidemiologic index and risk factors ( table 2) . two studies reported on the risk factors of mers infection. alraddadi et al. [42] analysed the effect of nonhuman contact, including travel history, animal-related exposure, food exposure, health condition, and behaviour and reported direct dromedary exposure, diabetes or heart disease, and smoking as risk factors of mers infection. another study reported older age, outbreak week, and nationality as risk factors [43] . three studies analysed factors associated with spreaders. non-isolated in-hospital days, hospitalization or emergency room visits before isolation, deceased patients, and clinical symptoms, including fever, chest x-ray abnormality in more than three lung zones, and the cycle threshold value, were related to spreaders [34, 44, 45] . four studies reported risk factors of mers severity. the included studies showed that the prnt 50 and cd4 t cell response [46] as well as a high mers virus load [47] were associated with the severity of mers. additionally, male sex; older age; concomitant disease, including hypertension; and symptoms, including fever, thrombocytopenia, lymphopenia, and low albumin concentration, were related to mers severity or secondary disease [47] [48] [49] . fifteen studies reported risk factors of mortality in mers patients. older age [4, 25, 32, [49] [50] [51] [52] [53] [54] [55] and comorbidity [29, [50] [51] [52] 54] , including diabetes [32, 55] , chronic kidney disease [32] , respiratory disease [4, 55] , pneumonia [56] , cardiac disease, and cancer [53] , were the most prevalent in the included studies. male sex was reported as a risk factor in one study [56] . smoking [32, 56] and location of acquisition [51, 53] were also reported. while one study noted that hcw, as a profession, was associated with mortality [53] , non-hcws were reported to be related to mortality in two other studies [50, 51] . additionally, a shorter incubation period [25, 56] , longer duration of symptoms [32] , more days from onset to confirmation [29] , later epidemic period [52] , and longer hospitalized days [29] were reported as mortality risk factors. symptoms at diagnosis, including abnormal renal function [56] , respiratory symptoms [56] , gastrointestinal symptoms [32] , lower blood pressure [32, 55] , and leucocytosis [55, 56] , were also found to be associated with mortality in mers patients. severity of illness, [50, 51] such as need for vasopressors [57] , chest radiographic score [58] , health condition [59] , use of mechanical ventilation [55] , and occurrence of dyspnoea [55] were also found to increase the mortality risk. the characteristics of mers differ between south korea and the middle east area. the r value of mers was reported to be below 1 in the middle east area, except in one study [15] , but was from 2.5-8.1 in south korea [15] [16] [17] [18] [19] . although studies using data from the middle east area reported 0.42-4% secondary attack rates, studies in south korea reported 4-6% secondary attack rates for patients or hospital visitors [5] , and 3.7-15.8% for the overall attack rate [23, 24] . the mers incubation period was reported to be 4.5-5.2 days in the middle east area [7, 29] , but this period was found to be slightly longer in south korea [3] [4] [5] [23] [24] [25] [26] [27] [28] . the severity of mers also differed between the middle east area and south korea. mortality of mers patients was found to be 20.4% in south korea based on a report including all cases [27] , but most studies from saudi arabia reported higher rates, from 22 to 69.2% [7, 22, 33, [37] [38] [39] . days from onset to confirmation were similar, 4-8 days in the middle east area [29, 31] and 4-6.5 days in south korea [3, 28, 29] . days from onset to discharge were slightly longer in south korea, 14-17 days in the middle east area [29, 36] and 17-20 days in south korea [3, 29] (table 3 ). the transmissibility and severity of mers were different by outbreak countries, especially between the middle east area and south korea. the virus, host, and environmental factors may be the causes of the mers outbreakrelated differences between the two regions. from the standpoint of viral factors, there was a mutation of the mers coronavirus (mers-cov) in the south korea outbreak. kim et al. [60] reported a point mutation in the receptor-binding domain of the viral spike protein in mers-cov, and another study showed that mers-cov in south korea had higher genetic variability and mutation rates [61] . individual characteristics can also affect mers transmission. as previous studies showed, there is an association between older age and mers infection [43] , severity [48] , and mortality [4, 50] , and the population structure may be related to transmission and severity. additionally, individuals aware of mers were found to be more likely to practice preventive behaviour [62] , which differed by demographic characteristics [63, 64] . the transmission environment may also contribute to the difference. while many mers cases were contracted through exposure to camels in saudi arabia [42] , the south korea outbreak involved multiple generations of secondary infections caused by intra-hospital and hospital-tohospital transmission [3, 65] . strategies considering various factors are therefore needed to assess the impact of mers and to better control its spread. although several studies have reported the overall r value [9, 10, 14, 19] , others have shown that this value this can be variable based on the generation or a control intervention [11, 16, 19] . especially in the south korea epidemic, the r value was particularly high in the early stage or first generation, at 4.42-5.4, though it later decreased to 0.14-0.39 [16, 19] . further studies should consider and analyse the variation of the r value depending on the period or control intervention. while earlier studies on infectious diseases assumed a homogeneous infection ability of a population, recent studies have shown the existence of so-called super spreaders, individuals with a high potential to infect others in many infectious diseases, including ebola and severe acute respiratory syndrome (sars) [66] . the role of the super spreader is also important in the spread of mers. in south korea, 83.2% of mers patients were associated with five super-spreading events [27] . stein et al. [67] asserted that super spreaders were related with the host, pathogen, and environmental factors, and wong et al. [66] reported that individual behaviours could also contribute to disease spread. there are variations in the mortality and attack rates among studies using south korea data. for example, park et al. [24] reported a 47.8% mers mortality, while reports from the korean ministry of health and welfare showed 20.4% mers mortality. this disparity may, in part, be due to small sample sizes. park et al. [24] included only 23 patients because the study was conducted in an early phase of a mers outbreak. we excluded studies that included cases with < 20 subjects, which were mostly case series, to reduce those types of biases. the present review found that older age and concomitant disease were risk factors of mers infection and mortality. these results are consistent with a recent systematic review that reported older age, male, and an underlying medical condition as predictors of death related to mers [68] ; therefore, these factors should be prioritized in protection and treatment procedures. one limitation of this study was the possibility of subject duplication. especially in south korea, the korean government publishes mers reports that include all patients. the epidemiologic index in other studies might be biased since they included partial korean patients and were analysed in the middle of an outbreak. however, we included those studies because they showed the characteristics of mers in different situations and different stages. we did not conduct a meta-analysis because of the small number of studies for each index, which might be another limitation of this study. although this study reviewed the risk factors of mers and their impact, assessing the effect size of each risk factor is important. more studies investigating the effect of risk factors on mers need to be constantly conducted. most studies on the transmissibility and severity of mers have originated from saudi arabia and south korea. even though the r 0 value in south korea was higher than that in saudi arabia, mortality was higher in saudi arabia. the most common factors behind mers infection and mortality were older age and concomitant disease. future studies should consider the risk of mers based on the outbreak region and patient characteristics. the results of the present study are valuable for informing further studies and health policy in preparation for mers outbreaks. isolation of a novel 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behaviours and risk perception among nursing students during outbreak probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea the role of super-spreaders in infectious disease super-spreaders in infectious diseases clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis authors' contributions jep (corresponding author) designed the study, and conducted the data search and the analysis with jep (1st author). syj and ark participated in the data review. jep (corresponding) drafted the manuscript, and jep (1st), syj, and ark revised it. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. the authors declare that they have no competing interests. key: cord-318585-cp76qr9f authors: matsuyama, ryota; nishiura, hiroshi; kutsuna, satoshi; hayakawa, kayoko; ohmagari, norio title: clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis date: 2016-11-29 journal: bmc public health doi: 10.1186/s12889-016-3881-4 sha: doc_id: 318585 cord_uid: cp76qr9f background: while the risk of severe complications of middle east respiratory syndrome (mers) and its determinants have been explored in previous studies, a systematic analysis of published articles with different designs and populations has yet to be conducted. the present study aimed to systematically review the risk of death associated with mers as well as risk factors for associated complications. methods: pubmed and web of science databases were searched for clinical and epidemiological studies on confirmed cases of mers. eligible articles reported clinical outcomes, especially severe complications or death associated with mers. risks of admission to intensive care unit (icu), mechanical ventilation and death were estimated. subsequently, potential associations between mers-associated death and age, sex, underlying medical conditions and study design were explored. results: a total of 25 eligible articles were identified. the case fatality risk ranged from 14.5 to 100%, with the pooled estimate at 39.1%. the risks of icu admission and mechanical ventilation ranged from 44.4 to 100% and from 25.0 to 100%, with pooled estimates at 78.2 and 73.0%, respectively. these risks showed a substantial heterogeneity among the identified studies, and appeared to be the highest in case studies focusing on icu cases. we identified older age, male sex and underlying medical conditions, including diabetes mellitus, renal disease, respiratory disease, heart disease and hypertension, as clinical predictors of death associated with mers. in icu case studies, the expected odds ratios (or) of death among patients with underlying heart disease or renal disease to patients without such comorbidities were 0.6 (95% confidence interval (ci): 0.1, 4.3) and 0.6 (95% ci: 0.0, 2.1), respectively, while the ors were 3.8 (95% ci: 3.4, 4.2) and 2.4 (95% ci: 2.0, 2.9), respectively, in studies with other types of designs. conclusions: the heterogeneity for the risk of death and severe manifestations was substantially high among the studies, and varying study designs was one of the underlying reasons for this heterogeneity. a statistical estimation of the risk of mers death and identification of risk factors must be conducted, particularly considering the study design and potential biases associated with case detection and diagnosis. electronic supplementary material: the online version of this article (doi:10.1186/s12889-016-3881-4) contains supplementary material, which is available to authorized users. cases of middle east respiratory syndrome (mers), caused by mers-associated coronavirus (mers-cov), have continuously been reported since june 2012. as of 29 june 2016, the total number of laboratory-confirmed cases notified to the world health organization (who) reached 1,768 cases, including 630 deaths [1] . particularly large outbreaks of mers-cov infection have been reported in the kingdom of saudi arabia (ksa) and the republic of korea (rok), while smaller outbreaks and importation events have been reported in other 25 countries [1] . of these, 10 countries are located in the middle east, 7 countries in europe, 3 countries in africa, 3 countries in southeast and east asia, and 1 in north america (the united states of america) [2] [3] [4] . because of the regular reporting of mers cases in the middle east, countries across the world are now facing a continuous threat of mers outbreak. to understand the clinical burden of mers, it is necessary to quantify the risk of developing severe clinical manifestations. the case fatality risk (cfr) is a measure of the risk of death among those who satisfy the case condition [5] , while risks of admission to an intensive care unit (icu) and that of requiring mechanical ventilation are also useful to measure the extent of developing severe mers complications. however, it is not only necessary to estimate such risks, but it is also critically important to identify epidemiological determinants of those risks to then predict the risk of severe complications for each patient before the onset of disease exacerbation [6] . in previous studies, the risk of death among secondary cases was estimated based on statistical modelling and was found to range from 20 to 22%, approximately [7] [8] [9] [10] [11] . meanwhile, among the primary cases, the risk of death was estimated to be greater at approximately 40%, perhaps because of biases associated with case detection and diagnosis [6] [7] [8] . as for epidemiological determinants of mers death, elderly patients with underlying comorbidities have been identified as the most susceptible population with a high risk of death [6, 9, 11] . despite our further understanding of the risk of developing severe mers, the abovementioned estimates are mostly based on a subset of mers cases; for instance, some of the risk estimates are a result of the analysis of cases diagnosed in 2015 in the rok or ksa alone. published articles with different study designs and populations have yielded different estimates and effect sizes associated with mers death. because of this variability, it is valuable to comprehensively and systematically analyze published mers studies that have recorded the clinical prognoses of cases. a systematic review is a highly informative review method that combines published results from different studies, thereby merging and contrasting results across multiple studies and answering study questions using the pooled estimates [12] . thus, we aimed to perform a systematic review to assess risks of death and other severe complications and determine the risk factors for mers-associated death and contrast these results by study population and study design. the present study was a systematic review conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [13] . pico statement: our study question is focused on laboratory confirmed cases of mers regardless of their treatment status, and thus, involves only retrospective observational studies, measuring their risks of admission to intensive care unit (icu) and death and comparing those risks by age, gender and underlying comorbidities. our systematic review protocol is summarized as additional file 1. published studies that referred to the clinical prognosis of mers cases were retrieved from medline (pubmed) and web of science electronic databases on 16 may 2016. the following search terms were used in "all fields" to identify relevant published articles: 1. "mers" or "middle east respiratory syndrome" or "novel coronavirus" or "novel coronavirus 2012" 2. "sever*"or "fatal*"or "death" or "mortalit*" 3. "hospitalization" or "intensive care" or "icu" 4. 1 and 2 and 3 we limited the search to articles published between april 2012 (i.e., after the first mers case was reported) and june 2016. additional studies reporting associated outcomes that were not identified by the abovementioned search strategy were manually retrieved by tracking the references of included articles (i.e., ancestry and discordancy approach). we restricted ourselves to publications written in english. all titles identified by the abovementioned search strategy were independently screened by two authors (rm and hn). abstracts of potentially relevant articles were subsequently reviewed for eligibility, and if a description of severe or lethal mers was available, articles were selected for closer examination of the full text. to be eligible for inclusion, published studies were required to meet the following characteristics: (i) studies focused on patients infected with mers-cov and (ii) explicitly documenting clinical outcomes (i.e., prognosis) and characteristics of both surviving and deceased patients. studies that allowed us to stratify the risk of severe or fatal mers by demographic or medical condition were preferred, but this was not an essential inclusion criterion. to calculate the risk of severe mers or mers death, we excluded case reports that documented only one or two cases (i.e., case reports with a sample size n ≥ 3 were eligible). included studies were further classified into five groups based on the study design and population studied: (i) case reports comprising published studies that described the clinical course of individual patients including mild cases; (ii) studies including only icu cases (hereafter referred to as icu studies): case reports or retrospective studies that reported outcomes of patients admitted to the icu only; (iii) hospital studies: retrospective or descriptive studies that aimed to document the outbreak in a hospital or healthcare-associated facility; (iv) retrospective studies: published studies that retrospectively analyzed the series of mers cases that were registered in the patient database or tracked medical records; and (v) surveillance studies: published studies that extracted data from a database of cases, systematically gathering epidemiological data, as coordinated by a country or who. the primary data extracted were the proportions of deceased mers patients, patients admitted to the icu and patients undergoing mechanical ventilation. all of these outcomes were dealt with as dichotomous variables, and thus, we calculated the 95% confidence interval (ci) for each included study using the binomial distribution. whenever possible, we stratified the risk of death by age, sex, underlying medical condition and study design. for the analysis of the effect of each covariate on the outcome, the odds ratio (or) for death among those with underlying conditions was calculated and compared with those without comorbidities. stratified analysis could not have been made for the proportions of icu admission and mechanical ventilation because the dataset of such covariates was not commonly available for these two outcomes. we employed a fixed effects inverse variance weighted model. weighted means (i.e. pooled estimate) of the abovementioned proportions and the or for death by each covariate were calculated using the inverse of variance estimates from each study. the heterogeneity among identified studies was statistically assessed by the i 2 statistic. to explore the possible sources of heterogeneity, we stratified pooled estimates by study design. a forest plot was used to illustrate the distribution of the outcome and effect size obtained from each published study. the flow diagram of the search and study selection process is shown in fig. 1 . among a total of 599 potentially relevant articles, 575 and 13 articles were excluded by screening of the titles and abstracts, respectively. one article was excluded by full-text screening. following the same process for 23 additional manually identified articles, a total of 25 articles were selected as eligible articles [8, and all were subject to meta-analysis. of these, four studies were classified as case reports, four as reports of icu cases, four as hospital outbreak studies, eight as retrospective studies and five as surveillance study. the majority of included articles were reported either from the ksa or the rok, except for one study conducted in jordan [22] and the who the estimated cfr was reported in 25 articles, ranging from 14.5 to 100% (fig. 2) . the pooled cfr was 39.1% (95% ci: 37.2, 41.1), but the i2 was as large as 92.4%. the sample size of case reports ranged from 3 to 12, while studies with other designs tended to have larger samples, with 10 or more cases, except for one icu study, one retrospective study and one hospital outbreak study. the proportions of icu admission and mechanical ventilation among all cases were available in 12 and 16 articles, respectively. the proportion of icu admission ranged from 44.4 to 100% with the pooled estimate at 78.2% (95% ci: 73.5, 82.9) and an i 2 value of 78.2%. the proportion of mechanical ventilation ranged from 25.0 to 100% with the pooled estimate at 73.0% (95% ci: 68.5, 77.5) and an i 2 value of 68.0%. [19] age and sex distributions are shown in relation to the risk of death by mers in fig. 3 . in the majority of the studies (except for a study from jordan), survivors were younger than those who died of mers. although not generally, infected men tended to die more often than women, and the pooled or of death among men compared with women was 1.4 (95% ci: 1.1, 1.6 ). the i 2 value of the sex difference for the risk of death was 48.6%. the risks of death, icu admission and mechanical ventilation were stratified by study design and are shown in fig. 4 , in which the pooled estimate for each study design was compared. the risk of death in the hospital outbreak and surveillance studies was significantly smaller than in icu case and retrospective studies. risks of icu admission and mechanical ventilation were the highest among icu case studies, followed by case report and retrospective studies. hospital outbreak studies yielded the smallest pooled risks of icu admission and mechanical ventilation. when comparing surveillancebased data between ksa and rok (fig. 2) , the risk of death in rok (i.e., 14.5-22.0% [8, 33, 34] ) tended to be lower than that in ksa (i.e., 46.0% by alsahafi and cheng [35] ), perhaps reflecting the presence of the contact tracing effort in the rok. figure 5 shows the possible association between five selected underlying medical conditions and the risk of death by mers. pooled estimates of the or were greater than the value of 1 for all five comorbidities, including diabetes mellitus (n = 8 studies), renal disease (6 studies), respiratory disease (5 studies), heart disease (5 studies) and hypertension (5 studies). among a total of five predictors, heart disease yielded the greatest or value at 3.5 (95% ci: 3.1, 4.8) followed by respiratory disease with an or of 3.1 (95% ci: 2.6, 4.2). figure 6 shows the potential association between the risk of death by mers and potential predictors, including sex, heart disease and renal disease. men from icu studies tended to yield a greater or for death compared with other study designs. conversely, expected values of ors for death among those with heart disease and renal disease compared with those without appeared to be lower than the value of 1.0. the present study systematically reviewed the risk of severe manifestations and death by mers by systematically searching and analyzing published articles from the ksa and the rok and calculating not only the cfr but [16] . icu represents intensive care unit also the risks of icu admission and requiring mechanical ventilation. several clinical predictors of death were identified including older age, male sex and underlying medical conditions, including diabetes mellitus, renal disease, respiratory disease, heart disease and hypertension. the risk estimate appeared to vary by study design. in particular, studies focusing on patients in the icu yielded the greatest estimates, while the cfrs for surveillance and hospital outbreak studies were smaller. these findings indicate that ascertainment biases in surveillance and hospital outbreak studies, frequently involving case finding effort, were smaller than in other types of studies. the importance of case finding effort is likely reflected in the different cfr estimates based on surveillance data between ksa and rok. although the presently identified clinical predictors are in line with previously published studies [6, 7, 11] , the present study is the first to systematically analyze published studies, including clinical research studies, and extract findings that echo those of published articles. as was observed in this study, systematic search and analysis of the transmission characteristics [38] and spatial spreading patterns of mers [39] have been successful. an important contribution of the present study is that we demonstrated that the risk of death or severe manifestations is highly heterogeneous for various reasons, including different study designs. it is recognized that mers involves asymptomatic infection [9] , and thus, studies must be clear as to how the risk is estimated, including the definition and diagnostic methods used to identify infected individuals. depending on the study design, the clinical predictors of death also differed. for fig. 4 estimated risks associated with middle east respiratory syndrome (mers) by study design. panels show the risk estimates by study outcome: (a) risk of death, (b) risk of admission to intensive care unit (icu) and (c) risk of mechanical ventilation. cfr represents the case fatality risk. the estimate for each study design represents the pooled risk of death calculated using the inverse variance of the risk of death in each published study. the size of the diamonds reflects the sample size, and the whiskers extend to the lower and upper values of the 95% confidence interval (ci). the diamond without fill represents the pooled estimate using the inverse variance of the risk of death. i 2 measures the extent of the heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity example, renal and heart diseases might not predict the risk of death in an icu setting, but they may be critically important in other settings that involve milder cases. not only studies in icu settings, but also retrospective studies yielded relatively high risk estimates for severe manifestations and death. our finding raises concerns regarding the retrospective analysis of confirmed cases in registered databases without referring to biases associated with case detection and diagnosis, which could yield a biased risk estimate of mers severity. in fact, that could explain why the cfr of confirmed cases among registered cases in patients' database has been as high as 40%, while the cfr of secondary cases in the presence of contact tracing has been estimated at about 20% [6] [7] [8] [9] [10] . the comorbidities identified in our study are in line with those already identified elsewhere [6, 37] . the identification of comorbidities is not only stressed based on previous and present findings [11] , but it is critically important to understand the underlying pathophysiological mechanisms. high representation of men among deceased cases may reflect the interaction of factors related to sex-specific lifestyle (e.g., smoking habits in the middle east). older age might reflect the greater likelihood of having underlying medical conditions. diabetes, renal and respiratory diseases could predispose patients to be immunologically vulnerable and heart disease could induce water retention (e.g., secondary aldosteronism), both exacerbating the systemic condition. hypertension could have been confounded by some other explanatory factor (s), for example, obesity could have likely led to both hypertension and mers death. nevertheless, identified predictors are accompanied by reasonable biological explanations. the present study is not free from limitations. the biggest concern is, given the absence of identifying information, the included articles most likely referred to the same cases multiple times, potentially overestimating the . the vertical dashed line shows the threshold value of or = 1. the diamond without fill represents the pooled estimate using the inverse variance of the or. i 2 measures the extent of heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity number of cases. in fact, the total number of diagnosed and reported cases of mers as of june 2016 is approximately 1,768 cases, but our systematic review included as many as 2,081 cases. thus, it is likely that multiple reports from rok (e.g., cowling et al. [8] , kcdcp [33] and majumder et al. [34] ) reported on the same cases multiple times. rather, we did not avoid any overlap of cases in datasets because that adjustment forced us to adjust the overlap among the cases from the ksa in a similar manner. for this reason, the pooled estimate would never represent the actual pooled outcome data because the same case was counted multiple times. if we remove cowling et al. [8] and majumder et al. [34] from our analysis and include kcdcp [33] , which had the largest sample size, the pooled estimate of the cfr would be increased to 45.4% (95% ci: 43.2, 47.7). this is understandable owing to the diminished impact of the extensive contact tracing effort in the rok. despite these overlaps, we conducted this systematic review to demonstrate that ascertainment biases likely act as a key factor that characterizes differential mortality across countries. to avoid any overlap of cases and better identify risk factors of icu admission and death, it is advised to set up a common case registration system across countries and allocate identity number for each individual case. as the second technical limitation to remember, it should be noted that the access to individual data was not achieved, and thus, for instance the age-related analysis did not rest on individual age data, and similarly, we have had limitations in the precision of the majority of outcome evaluations. third, clinical predictors of death have been classified only at organ level, and moreover, individual behavioral factors or habitat [40] have not been examined in relation to the risk of mers death. fourth, non-english language manuscripts have been missed, and they include at least a few publications in korea and one from jordan. despite these problems, we cannot help but consider that the present study successfully and systematically . odds ratio (or) represents the odds ratio of death among men with underlying medical condition compared with women without comorbidities, respectively. the size of the diamonds reflects the sample size, and the whiskers extend to the lower and upper values of the 95% confidence interval (ci). the diamond without fill represents the pooled estimate using the inverse variance of the risk of death. i 2 measures the extent of heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity evaluated the risk of severe manifestations and death by mers by collecting published information on clinical predictors of the risk of death. an important consideration is that the associated risk estimation and identification of risk factors of mers call for particular care in terms of study design, especially in aiming to eliminate biases associated with detection and diagnosis. heterogeneity in risks of death and severe manifestations secondary to mers was substantial. differential study design was one of underlying reasons for the large heterogeneity. statistical estimation of the risk of mers death and identification of risk factors must be conducted with particular careful attention paid to study design, especially accounting for biases associated with case detection and diagnosis. additional file middle east respiratory syndrome coronavirus (mers-cov). geneva: world health organization clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus (mers-cov) into the united states first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission case fatality: rate, ratio, or risk? middle east respiratory syndrome middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility preliminary epidemiologic assessment of mers-cov outbreak in south korea estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea systematic reviews and meta analysis of published studies: an overview and best practices preferred reporting items for systematic reviews and meta-analyses: the prisma statement a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case family cluster of middle east respiratory syndrome coronavirus infections ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological investigation of mers-cov spread in a single hospital in south korea acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study association of higher mers-cov virus load with severe disease and death, saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus outbreak in the republic of korea mortality risk factors for middle east respiratory syndrome outbreak, south korea the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study hospital outbreak of middle east respiratory syndrome coronavirus risk of mers importation and onward transmission: a systematic review and analysis of cases reported to who predicting the international spread of middle east respiratory syndrome (mers) identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, 2015: a retrospective epidemiological analysis funding hn received funding support from the japan agency for medical research and development, the japanese society for the promotion of science (jsps) kakenhi grant numbers 16kt0130, 16 k15356 and 26700028, the japan science and technology agency (jst) crest program and ristex program for science of science, technology and innovation policy. no received funding support from the ministry of health, labor, and welfare, japan (h27-shinkogyosei -shitei-006). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. collected datasheet is available from the authors (rm) upon request. authors' contributions hn conceived the systematic review. rm and hn implemented systematic search. rm and hn performed statistical analyses. rm and hn drafted the early version of the manuscript and hn substantially rewrote the text. sk, kh and no further revised the manuscript. all other authors gave comments on the revised manuscript and approved the final version of the manuscript. the authors are experts with interest in infectious disease epidemiology and also in clinical infectious diseases, and the team of lead author is led by professor from hokkaido university graduate school of medicine. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable.author details 1 graduate school of medicine, hokkaido university, kita 15 jo nishi 7 chome, kita-ku, sapporo 060-8638, japan. 2 crest, japan science and technology agency, 4-1-8, honcho, kawaguchi-shi, saitama 332-0012, japan.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-345081-15s2i6f0 authors: al-sehaibany, fares s. title: middle east respiratory syndrome in children: dental considerations date: 2017-04-17 journal: saudi med j doi: 10.15537/smj.2017.4.15777 sha: doc_id: 345081 cord_uid: 15s2i6f0 as of january 2016, 1,633 laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection and 587 mers-related deaths have been reported by the world health organization globally. middle east respiratory syndrome coronavirus may occur sporadically in communities or may be transmitted within families or hospitals. the number of confirmed mers-cov cases among healthcare workers has been increasing. middle east respiratory syndrome coronavirus may also spread through aerosols generated during various dental treatments, resulting in transmission between patients and dentists. as mers-cov cases have also been reported among children, pediatric dentists are at risk of mers-cov infection. this review discusses mers-cov infection in children and healthcare workers, especially pediatric dentists, and considerations pertaining to pediatric dentistry. although no cases of mers-cov transmission between a patient and a dentist have yet been reported, the risk of mers-cov transmission from an infected patient may be high due to the unique work environment of dentists (aerosol generation). middle east respiratory syndrome coronavirus is a novel beta coronavirus of the coronaviridae family that causes a severe respiratory disease with a high fatality rate. [3] [4] [5] [6] as of january 2016, 1633 laboratoryconfirmed cases of mers-cov infection and 587 related deaths have been reported by the world health organization (who) globally. 7 the male-to-female ratio of the affected patients was 2:1, with a median age of 49 years. 8 the incubation period for human-tohuman transmission ranges from 2-15 days. 9 the symptoms of mers-cov infection range from being asymptomatic to severe pneumonia, acute respiratory distress syndrome, septic shock, and multi-organ failure, leading to death. the symptomatic patients may have fever, cough, chills, throat soreness, myalgia, arthralgia, vomiting, or diarrhea. 10 furthermore, men over 45 years of age, patients with comorbidities, and healthcare workers have been reported to be high-risk groups for mers-cov infection. 11 the virus has been detected in respiratory, gastrointestinal and other bodily secretions, 12, 13 as well as in air samples, which indicates the possibility for airborne transmission. 14 several studies have reported mers-cov infections among healthcare workers 4,6,15,16 and children. 1, 17 viral infections, such as severe acute respiratory syndrome saudi med j 2017; vol. 38 (4) www.smj.org.sa (sars-cov), may be transmitted to healthcare workers from infected patients through aerosols. 18 considering that several types of dental equipment, such as handpieces, air-water syringes and ultrasonic scalers, produce considerable amounts of aerosols, the potential for the spread of infections from patients to dentists or dental assistants is high. 19 this review is an attempt to discuss mers-cov infection among children and those providing dental treatment to them, including precautions and considerations pertaining to the practice of pediatric dentistry. mers-cov infection in healthcare workers. the 3 patterns of mers-cov transmission suggested in the literature include the following: a) sporadic cases occurring in communities; b) transmission within families; and c) nosocomial transmission. 20 healthcarerelated mers-cov transmission is associated with high morbidity, extended use of mechanical ventilation and fatality rates of up to 65%; this type of transmission can be attributed to shortcomings in observing stringent infection control protocols. 4, 6, 15, 21 furthermore, mers-cov has been reported to be viable in hospitallike environments for up to 48 hours with a stability that is unaltered during aerosolization. 22 among 200 healthcare workers' contacts identified in al-hassa, saudi arabia, mers-cov infection was confirmed in 2 cases. 4 a saudi study 20 compared healthcare worker and family contact with laboratory-confirmed mers-cov patients and reported a lower rate (1.12%) of infection among the healthcare workers than among the families (3.4%). healthcare workers could be infected with mers-cov through exposure in the community or at their workplace, 1,6,20 they could be diagnosed late, 1,6 and they could remain asymptomatic or mildly symptomatic. 6, 8, 16 furthermore, unsuspected cases entering healthcare facilities have been considered the main source of mers-cov. 4 considering these aspects and that healthcare workers may continue to work regardless of being symptomatic, 15 the possibility of transmitting the infection to their patients is also high. memish et al 1 reported that the age of 11 saudi pediatric patients who presented clinically with symptoms of mers-cov infection ranged from 2-16 years with a female-to-male ratio of 2.7 to 1. of the 11 cases, 2 were symptomatic, and 9 were asymptomatic with no underlying comorbidities. another report by thabet et al 17 concluded that although mers-cov infection presents with a wide range of clinical manifestations, the mortality rate in children is lower than that in adults. a review on the effects of coronavirus infection in children concluded that human coronavirus infections may be associated with respiratory symptoms and may also involve central nervous system. the authors suggested that the clinical and genetic traits of human coronavirus infection among children should be monitored closely for early prevention. 23 dental considerations. bioaerosols in dental practice. bioaerosols are defined as a suspension of biological particles in gaseous media. 24 apart from radiation exposures, dermatitis, eye injuries, and musculoskeletal and respiratory disorders, other occupational health risks exist in dentistry. these risks include percutaneous exposure incidents and exposure to infectious diseases, such as those that may be present in bioaerosols. 25 subgingival scaling of periodontally involved teeth with ultrasonic scalers may produce aerosols containing blood. 26 one study reported that ultrasonic scalers and tips produced significantly more aerosol and splatter than a handheld curette, regardless of the scaler type used. 27 miller et al 28 studied the characteristics of blood-containing aerosols generated by common powered dental instruments. the author concluded that all the recovered particles could contain the hepatitis b virus and be inhaled and retained (20-100%) in the human respiratory system. the repeated and chronic exposure to bioaerosols generated during certain dental procedures and the relatively small particle size contribute to an increased risk of infection among dental professionals. 29 furthermore, the protection provided by surgical masks worn by dental professionals may be low for small particles; in addition, these masks may not fit perfectly in clinical practice. 30 a study reported that 15-83% of plasma aerosol particles ranging from 0.06-2.5 microns in size passed through the filters of 9 makes of surgical masks used by dentists. 28 these factors may play a role in the airborne route of viral infections among dentists and clinical dental auxiliaries. the likelihood of detecting, reporting, documenting and publishing dentistry-associated infections is relatively less. 31 although no definitive evidence of an extensive public health hazard from exposure to dental unit waterlines has been reported. 32 few case reports have suggested a definitive link between exposure to contaminated dental unit waterlines and legionella infection. 33, 34 disclosure. authors have no conflict of interest, and the work was not supported or funded by any drug company. infection control in dental practice. a review by scully and samaranayake 35 on the emerging and fluctuating viral diseases in the new millennium concluded that infection control plays an equally important role in the practice of dentistry as do the understanding of oral manifestations and the diagnosis and management of viral infections. while differences exist in the virology, epidemiology, and clinical outcomes of mers-cov and sars-cov infections, 36 the clinical symptoms of mers-cov resemble those of sars-cov, apart from acute renal failure. 37 although these results cannot be directly interpreted for a definitive conclusion, the management protocols for different mers-cov infection-associated with clinical scenarios for dental professionals may be similar to those of sars-cov. the implications of sars-cov for general dental practitioners, the significance of droplets and aerosols in disease transmission, the recommended management protocols for sars-cov infection and specific infection control measures have been well described by li et al. 38 a comprehensive review on universal and standard precautions has been published elsewhere 39 and is beyond the scope of this article, infection control measures pertaining only to pediatric dental practice in the context of mers-cov infection are discussed here. in pediatric dental practice, effective infection control measures for the prevention or minimization of viral infection transmission can be implemented by a) controlling the gag or cough reflex; b) reducing aerosol/ splatter generation; c) managing contaminated air and; d) improving personal protection. the gag or cough reflex may be stimulated by certain procedures, such as posterior intraoral and bite-wing radiographs and taking impressions. orthopantomographs or oblique lateral views may be considered instead of intraoral radiographs for screening, 40 whereas oral mucosa in very sensitive patients may be anesthetized before taking impressions. 41 sedation may also be considered to control gag reflex. 42 varying levels of physical, intellectual, emotional and social development of children and adolescents are major challenges for dentists especially attending to their psychological needs within adequate infection control regimen. toys provided for pediatric patients may be a potential source of cross-infection. soft toys are more likely to be contaminated, difficult to disinfect and may re-contaminate quickly compared to hardsurfaced toys. furthermore, restraining devices used to control movements of pediatric patients such as velcro fasteners may also be contaminated and should be disinfected accordingly. 43 extra-oral evacuation devices and special aerosol reduction devices may be used in combination with ultrasonic scalers to reduce the amount of aerosol produced. 44, 45 in high-risk cases, chemomechanical caries removal 46 or the atraumatic restorative technique 47, 48 may be utilized to prevent or reduce aerosol and splatter generation. in addition, high-volume evacuation removes infectious droplets at the source as they are emitted; thereby, minimizing, or preventing their dispersion in the air. to maintain their efficacy, the filters in the suction apparatus should be cleaned every day, and the exhaust air should be vented outside to prevent the recirculation of contaminated air. 38 contaminated air can be managed by improving dental clinic ventilation and/or by disinfecting the air. an ideal airflow pattern combined with a minimum of 3 air changes per hour has been recommended for dental clinics. [49] [50] [51] moreover, although its use in dental clinics is unconfirmed, ultraviolet germicidal irradiation may be installed and is effective against fungi, viruses, and bacteria, namely, tubercle bacilli and anthrax. 38, 49 on the other hand, measures of improving personal protection include washing hands frequently before and after treatment, using disposable barriers, dispensing instruments and materials just before treatment (thereby preventing particles from settling on the surfaces), and sterilizing soiled instruments. 38 after each patient visit, surfaces may be disinfected using hospital-grade disinfectants, which are effective against coronavirus. 52 personal protective equipment, such as gowns, hair covers, masks, gloves, shielded face masks and shoe covers, should be used as appropriate. 51 a higher level of respiratory protection should be considered, especially with aerosol-generating procedures. the use of n95 respirators offers a certain level of protection against the airborne transmission of sars-cov, although the exact level of protection offered by these respirators for individuals may vary. 53 examples of personal protective equipment such as face mask with plastic shield (figure 1 ) and n95 respirator (figure 2) . these respirators may also provide some level of protection against mers-cov and may be utilized instead of surgical masks in dental clinics. apart from these measures, vaccination of pediatric dentists and staffs working in pediatric dental clinics against measles, mumps, varicella and rubella may be necessary for their protection from these viral infections. 54 in conclusions, considering the unique work environment of dentists, which involves close patient contact and aerosol production, the risk of mers-cov transmission from an infected patient is high. children are also prone to mers-cov infection. as the number of mers-cov cases may increase in future, pediatric dentists should be well informed and educated about not only the signs and symptoms of the condition but also how to follow stringent infection control measures in these cases. middle east respiratory syndrome coronavirus disease in children isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov). update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of middle east respiratory syndrome (mers): a new zoonotic viral pneumonia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mers-cov outbreak in jeddah--a link to health care facilities a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome middle east respiratory syndrome coronavirus in children factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises aerosols and splatter in dentistry: a brief review of the literature and infection control implications screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions effects of coronavirus infections in children cultivation of bacteria and fungi occupational health problems in modern dentistry: a review blood contamination of the aerosols produced by in vivo use of ultrasonic scalers aerosol and splatter contamination from the operative site during ultrasonic scaling characteristics of blood-containing aerosols generated by common powered dental instruments measurement of airborne bacteria and endotoxin generated during dental cleaning efficacy of three face masks in preventing inhalation of airborne contaminants in dental practice infected health care workers and patient safety: a double standard risk assessment of dental unit waterline contamination a survey of methods used to detect nosocomial legionellosis among participants in the national nosocomial infections surveillance system legionella contamination of dental-unit water emerging and changing viral diseases in the new millennium middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection severe acute respiratory syndrome (sars) and the gdp. part ii: implications for gdps guidelines for infection control in dental health-care settings essentials of dental radiography and radiography oral bioscience. london (uk): churchill-livingstone sedation in dentistry. part 2: management of the gagging patient infection control issues related to pediatric dentistry reduction of bacteria-containing spray produced during ultrasonic scaling the usefulness of the modified extra-oral vacuum aspirator (eova) from household vacuum cleaner in reducing bacteria in dental aerosols chemochemical caries removal: a review of the techniques and latest developments the atraumatic restorative treatment (art) technique: does it have a place in everyday practice? atraumatic restorative treatment (art): rationale, technique, and development the application of ultraviolet germicidal irradiation to control transmission of airborne disease: bioterrorism countermeasure infection control for the dental team. copenhagen: munksgaard additional precautions for tuberculosis and a self-assessment checklist managing sars amidst uncertainty possible sars coronavirus transmission during cardiopulmonary resuscitation safety management and infection control in pediatric dentistry key: cord-256784-wfaqim7d authors: modjarrad, kayvon title: mers-cov vaccine candidates in development: the current landscape date: 2016-06-03 journal: vaccine doi: 10.1016/j.vaccine.2016.03.104 sha: doc_id: 256784 cord_uid: wfaqim7d middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than 1600 people, resulting in more than 600 deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of mers-cov have created an urgent need for effective public health countermeasures, paramount of which is an effective means of prevention through a vaccine or antibody prophylaxis. despite the relatively few number of cases to-date, research and development of mers-cov vaccine candidates is advancing quickly. this review surveys the landscape of these efforts across multiple groups in academia, government and industry. middle east respiratory syndrome (mers-cov) was first isolated in september 2012 from a patient in saudi arabia who presented two months earlier with severe acute respiratory infection and acute renal failure [1] . retrospective testing of samples in jordan identified earlier cases from a nosocomial outbreak in april 2012 [2] . although the majority of mers-cov cases (∼75%) have occurred in saudi arabia, 25 other countries have confirmed imported or autochthonously transmitted cases ( fig. 1) [3, 4] . the most recent and largest outbreak outside of saudi arabia occurred in south korea in may 2015 [5] , raising concern for an eruption of regional outbreaks or accelerated global spread, similar to the phylogenetically related severe acute respiratory syndrome coronavirus (sars-cov) that killed nearly a thousand people a decade earlier [6] . although the definitive host for mers-cov has not yet been established, closely related coronaviruses have been isolated from bats across wide geographic areas [7] [8] [9] . mounting evidence has strongly implicated dromedary camels as the intermediate animal reservoir, as serological surveys throughout the middle east and north africa have demonstrated them to have a high prevalence of mers-cov binding or neutralizing abs [10] [11] [12] [13] . additionally, outbreak investigations have suggested epidemiologic linkage between farm camels and human cases [14] . mers-cov is a spherical, enveloped, single-stranded, positive sense rna beta-coronavirus [1, 15] . its genome contains a replicase * tel.: +13015003600. locus at the 5 end and codes for structural proteins toward the 3 end. the most immunogenic of the viral proteins is spike (s), a trimeric, envelope-anchored, type i fusion glycoprotein that interfaces with its human host cognate receptor, dipeptidyl peptidase 4 (dpp4), to mediate viral entry [16, 17] . s comprises two subunits: s1, which contains the receptor-binding domain and determines cell tropism; and s2, the location of the cell fusion machinery. although dpp4 has a broad tissue distribution, most of the clinical manifestations of mers-cov can be attributed to its localization to the lower respiratory tract [18, 19] . much like other coronaviruses, mers-cov can also cause significant dysfunction of the gastrointestinal, cardiovascular, renal, and neurologic systems. mers-cov is distinct, though, in its tendency to cause greatest harm to older individuals with concurrent comorbidities of one or more of these organ-systems [20, 21] . despite past efforts to develop coronavirus countermeasures in response to the sars-cov pandemic, there are currently no prophylactic or therapeutic interventions of proven efficacy for mers-cov or any other coronavirus infection. although combination treatment with ribavirin and interferons were shown to improve clinical outcomes in mers-cov-infected non-human primates (nhps), treatment was initiated very soon after viral challenge (∼8 h) and results have not been replicated in humans [22] . in fact, no experimental interventions have demonstrated appreciable benefit in acutely ill patients in a consistent or controlled manner. rapidly scaled treatments based on naturally occurring neutralizing antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have demonstrated mortality reductions for other respiratory infections and may hold promise for mers-cov as well [23] . their development, however, is limited by logistical challenges, local technical capacity, and donor supply. supportive management, adapted from guidelines developed for sars-cov, has thus far been the mainstay of mers-cov treatment. the global will to develop a coronavirus vaccine faded in the aftermath of sars-cov pandemic but has since gained renewed momentum in the face of the current mers-cov outbreak. previous approaches to coronavirus vaccine development were broad and included whole-inactivated and live-attenuated viruses, recombinant vectors and protein subunits, as well as dna and rna based platforms [6] . most developers based their immunogen designs on the s surface glycoprotein, the primary target for neutralizing antibodies during any natural coronavirus infection. a number of preclinical and clinical studies showed that the sars-cov s1 protein subunit, and specifically the rbd at its core, could serve as a dominant target for neutralizing antibodies in mice, non-human primates, and humans [24] . s1, therefore, became the basis for a number of promising sars-cov vaccine candidates. the s1 protein subunit and rbd have also been the basis for several mers-cov vaccine candidates (fig. 2 ) [25] [26] [27] [28] [29] . resolution of rbd crystal structures alone or in complex with the dpp4 receptor [25, 30, 31] have informed the design of immunogens that have been expressed either as recombinant protein fragments or conjugates to the fragment crystallizable (fc) region of human antibodies. both types of constructs, in formulation with aluminum salt or oil-in-water adjuvants, have elicited neutralizing antibodies of high potency across multiple viral strains. despite their demonstrated immunogenicity in animal models and anticipated safety in humans, rbd or s1-subunit based vaccine candidates are limited in their epitope breadth. although the coronavirus genomes are not as variable as other rna viruses, the rbd is the most mutable region, containing mutation sites that define antibody escape variants [25, 32] . thus, vaccine candidates that elicit a more diverse antibody repertoire as well as a robust cellular immune response may offer the advantage of broader and more durable protection. full-length s used as an immunogen could at least increase the breadth of the antibody response; however, it has been difficult to express, and may require additional work to produce a stable soluble trimer of the s ectodomain. investigators at the university of maryland, in collaboration with novavax, inc., have overcome this problem through the development of s rosettes that are stable and immunogenic in murine models [33] . vaccines that mimic natural infection, such as live-attenuated viruses or recombinant viral vectors, may elicit even more robust immunity. live attenuated viruses have historically been among the most immunogenic platforms available, as they have the capacity to present multiple antigens across the viral life cycle in their native conformations. although a live-attenuated mers-cov has yet to be tested, one has been constructed and has the potential to be protective [34] . however, manufacturing live-attenuated viruses requires containment in a biosafety level 3 or 4 facility. additionally, live-attenuated viruses carry the hazards of inadequate attenuation or reversion to wild type form and causing disseminated disease, particularly in immunocompromised hosts. given that moderately immunocompromised adults with co-morbidities such as diabetes mellitus and chronic kidney disease have suffered the most severe mers-cov disease, these individuals may comprise a target population for immunization, thus making a live-attenuated virus vaccine a less viable option. replication competent viral vectors could pose a similar threat for disseminated disease in the immunosuppressed. replication deficient vectors, however, avoid that risk while maintaining the advantages of native antigen presentation, elicitation of t cell immunity and the ability to express multiple antigens. to date, two recombinant vector platforms-modified although replication deficient vectors are relatively safe and immunogenic, their ability to deliver genetic material for expression could be impeded by pre-existing or developing immunity to the vector itself. one way to overcome this limitation is by administering different vectors in a so-called prime-boost immunization regimen. as this strategy has been effective for other pathogens, it is likely that the same success could be recapitulated for mers-cov. the use of more than one type of platform or antigen in a single vaccine also increases the likelihood of inducing a broad repertoire of antibodies with diverse mechanisms of viral neutralization. one vaccine regimen developed at the us national institutes of health is based on full-length s dna and a truncated s1 subunit glycoprotein and has elicited neutralizing antibodies in mice directed at both the s1-within and outside the rbd-and s2 subunits. immunization with these constructs also protected nhps from severe lung disease after intra-tracheal challenge with mers-cov [25] . a dnaonly vaccine, expressing multiple antigens, has also been developed by inovio pharmaceuticals and geneone life science inc. and has been advanced to a phase i first-in-human trial. each of the vaccine candidates that have been mentioned is being developed for prophylactic use. however, as the total number of cases (>1600) [3, 4] and reproductive rate (∼0.7) of mers-cov are both relatively low [39] , it will be difficult to define the target populations for vaccination that would support the investment in manufacturing and advanced product development. also, with such low incidence and lack of robust animal models, it would be difficult to achieve a vaccine efficacy result that would be sufficient to support licensure. human mabs, on the other hand, could be used without as much discrimination in an outbreak setting for post-exposure prophylaxis and early treatment. the advantages of mabs over polyclonal antibodies (administered through convalescent plasma or hyperimmune globulin) are their higher potency, greater specificity, more extensive pre-licensing evaluation and consequently improved safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic level detail for the design of better immunogens. however, the timeline for mab development may be longer and potentially cost more than some vaccines. despite requirements for greater upfront investment, several groups have developed highly potent mabs that are currently being advanced through pre-clinical stages of testing (fig. 3) . some have been isolated from immunized animals (mice/humanized mice/nhps) [25, 40] , while others have been identified from either an antibody human phage library [41] [42] [43] [44] or memory b cells of infected and recovered human survivors [45] . almost all of the mabs that have been reported target the spike rbd. it is likely that mabs directed at other sites on the spike glycoprotein have been recovered but are not as potent neutralizers. most of those that have been published bind to recombinant spike with picomolar affinity and neutralize mers-cov pseudovirus at a half maximal inhibitory concentration (ic 50 ) of 0.1 mcg/l or less. additionally, some have demonstrated protective efficacy in pre-and post-exposure prophylaxis animal models [44, 45] . the successes thus far in isolating potent and protective mabs may prove useful for therapeutic development where the target population is well defined. it may prove more challenging to advancing these products to licensure and fullscale production at affordable costs for the purpose of prophylaxis in as of yet undefined populations. the vaguely defined epidemiology of mers-cov has complicated the design and implementation of appropriate public health countermeasures. most transmission events have occurred either in the setting of household clusters or nosocomial outbreaks [46] [47] [48] [49] [50] [51] . it is also likely that the virus has been introduced multiple times into human populations from a large zoonotic reservoir, i.e. dromedary camels. given the broad distribution and ownership of camels in the arabian peninsula where most cases have occurred, a targeted vaccine campaign may prove difficult. as the outbreak in the republic of korea revealed, patients and workers in the same healthcare facility as an infected patient are at high risk for secondary acquisition. an optimal strategy may be to use vaccines in conjunction with stringent infection control practices in hospitals where mers-cov cases are being treated. the epidemiologic link of mers-cov between bats, camels, and humans presents an opportunity for a veterinary vaccine to interrupt the transmission cycle. a successful precedent for this so-called "onehealth" approach toward mitigating human disease with a veterinary vaccine exists in the example of equivac ® , a hendra virus vaccine developed solely for horses [52] . although hendra virus is even more rare than mers-cov, it is highly fatal with no treatment other than intensive supportive management. in 2012, a protein subunit vaccine was licensed and rolled-out in australia, where all outbreaks of the virus occurred. since that time, the incidence in horses has fallen precipitously and no human cases have been detected [53] . a similar strategy may be applicable to mers-cov; however, a veterinary vaccination in this context would be deployed solely for the sake of protecting humans, as the virus causes only mild upper respiratory illness in camels. safety and reduction in viral shedding would have to be demonstrated in immunization, challenge and transmission studies of camel or camelid populations, one of which has shown efficacy [54] . one of the primary challenges to developing countermeasures to mers-cov is the lack of an appropriate animal model that recapitulates the natural history of human disease. much of the difficulty originates from the absence of the virus's cognate dpp4 receptor. one group approached this problem by successfully transducing mice with an adenoviral vector expressing human dpp4 [55] . although more relevant than a standard murine model, transient transduction of the desired protein may result in inconsistent tissue expression of adenovirus antigens. agrawal et al. made an important advance with the development of a transgenic mouse model that demonstrated productive, disseminated mers-cov infection [56] . although rhesus macaques do not manifest full clinical disease, they develop a transient lower respiratory infection that can be quantified and evaluated by computed tomography. investigators at the nih rocky mountain laboratories (rml) and integrated research facility (irf) have also independently been developing potentially lethal marmoset models that could be used for the evaluation of vaccines, mabs and therapeutics [57] . as mers-cov vaccines-both active and passive-are developed and tested, not only will more relevant animal models be required, but there will also be a need for a more detailed understanding of the epidemiology, immunology, and pathogenesis of the virus. in the aftermath of the west african ebola virus epidemic and in the face of the current zika virus outbreak, the global health community has coalesced around the realization that a multi-faceted plan is required to quickly and efficiently respond to global public health emergencies. the world health organization is currently developing a blueprint by which that preparation and response can follow, with mers-cov highlighted as a case study. although mers-cov still causes relatively few cases in a limited geographic distribution, its high case fatality and sudden outbreak in in korea have proven it to be a pathogen of public health concern. the concentration of the epidemic to saudi arabia also raises the specter of international spread every year during hajj, one of the largest mass gathering events in the world. ultimately, the development of a safe and effective vaccine for mers-cov may not yield its greatest benefit for the current epidemic but for the knowledge gained in creating a platform for combating coronaviruses as a whole. the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological findings from a retrospective investigation east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 11 update: middle east 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neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution preand postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus family cluster of middle east respiratory syndrome coronavirus infections hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections hendra virus vaccine, a one health approach to protecting horse, human, and environmental health hendra virus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection key: cord-265666-27ckjl7w authors: kang, hee sun; son, ye dong; chae, sun‐mi; corte, colleen title: working experiences of nurses during the middle east respiratory syndrome outbreak date: 2018-05-30 journal: int j nurs pract doi: 10.1111/ijn.12664 sha: doc_id: 265666 cord_uid: 27ckjl7w aims: to explore working experiences of nurses during middle east respiratory syndrome outbreak. background: since the first case of middle east respiratory syndrome was reported on may 20, 2015 in south korea, 186 people, including health care workers, were infected, and 36 died. design: a qualitative descriptive study. methods: seven focus groups and 3 individual in‐depth interviews were conducted from august to december 2015. content analysis was used. results: the following 4 major themes emerged: “experiencing burnout owing to the heavy workload,” “relying on personal protective equipment for safety,” “being busy with catching up with the new guidelines related to middle east respiratory syndrome,” and “caring for suspected or infected patients with caution.” participants experienced burnout because of the high volume of work and expressed safety concerns about being infected. unclear and frequently changing guidelines were 1 of the common causes of confusion. participants expressed that they need to be supported while caring for suspected or infected patients. conclusion: this study showed that creating a supportive and safe work environment is essential by ensuring adequate nurse staffing, supplying best‐quality personal protective equipment, and improving communication to provide the quality of care during infection outbreak. information on the new guidelines and job-related information via text messages using smartphones was helpful for the nurses. • creating a supportive work environment and providing adequate training for nurses is essential. the implications of this paper: • nurse managers and hospital administrators should establish strategies to prevent nurses from burnout and to ensure their safety during the outbreak of infectious diseases. • clear and consistent practice guidelines and effective communication methods among nurses should be developed. • increasing awareness of health care workers about infectious diseases to enhance emergency preparedness is essential. with mers on may 20, 2015, which was 9 days after his first visit (lee & ki, 2015; yang et al., 2015) . a mers outbreak in korea was caused by hospital-to-hospital transmission because patients were moved to other hospitals without appropriate quarantine (ki, 2015; kim et al., 2016) . it was exacerbated by overcrowding in the emergency room, delayed diagnosis, and lack of self-protection (balkhy, perl, & arabi, 2016; xia, zhang, xue, sun, & jin, 2015) . as more and more mers cases were reported, hospitals restricted the visitors and checked all visitors and employees for the presence of fever. additionally, for the temporary screening of mers-suspected cases, triage was set up to screen the infected or suspicious patients and to block the cross-transmission in and outside hospitals. furthermore, the government adopted the national safe hospital program to control mers infections within hospitals (korea centers for disease control and prevention, 2015 along with high risk of being infected, studies reported that health care personnel experienced occupational risks, distress, and the fear of contacting and transmitting the disease during epidemics of h1n1, severe acute respiratory syndrome (sars), and ebola virus (bukhari et al., 2016; chou et al., 2010; corley, hammond, & fraser, 2010; koh, hegney, & drury, 2012; speroni, seibert, & mallinson, 2015) . nurses also reported positive experiences of becoming more confident, mature, and broad-minded while caring for sars patients (liu & liehr, 2009) and positive feelings about their experience of caring for h1n1 patients (honey & wang, 2013) . however, few studies have been conducted on nurses' working experiences during the mers outbreak. the aim of the study was to explore the working experiences of nurses during the mers outbreak. data were collected using 7 focus group interviews and 3 individual in-depth interviews from august to december 2015 until the data were saturated. focus group questions were developed based on a literature review (chou et al., 2010; corley et al., 2010) . each focus group was comprised of 2 to 5 participants. individual in-depth interviews were conducted for those who were not able to meet in focus groups because of time conflicts. prior to the interview, participants were informed about the reasons for doing this study and the goals of the study. the first author (hsk), who has experience with qualitative research, conducted the focus groups and the individual in-depth interviews. the focus group discussions and individual interviews were conducted in a private room at a site with convenient participant access. each session lasted for 1 to 2 hours. no one was present besides the participants and the researchers during the interviews. a semistructured interview guide was used. we conducted a pilot test with 2 nurses caring for the patients with mers and refined the interview questions. the following questions guided the interviews: • what are your working experiences of caring for suspected or infected patients with mers during the outbreak? • what are the challenges of working during the mers outbreak? to ensure consistency and accuracy of our data, interviews were audio-taped with the participants' permission and transcribed verbatim. the researchers made field notes during and right after the • it's so sweaty and hard to breathe with it. it is hard to work and see clearly while wearing it (protective measures) and i feel dizzy when wearing it for long hours. • (we were) sweating, (find it) hard to breathe; it was difficult to work wearing personal protective equipment. being busy with catching up with the new guidelines for mers frequently changing guidelines • mers guidelines kept changing. at first, (we were told to) do thing this way and this is the guideline. we needed time to understand and practice a new guideline; however, guidelines kept changing without considering our adjustment to a new one. • the most difficult thing was that protocols were changed daily. working along with memorizing new protocols was very difficult. while workload increased, (we) were told this has been changed this way and that has been changed that way in shift change meetings. sharing the new information • we promptly communicated and shared updated information among nurses within the unit, through kakao talk (a free mobile instant messaging application for smartphones with free texting). • we had a notice note summarized about new information on mers. when changing shifts, we read the note and were also told what we have to be cautious because of what has been changed and it helped. it helped because we never had mers before and didn't know how we have to send the specimens and did not know how to cope with it. it worked as basic guidelines. lack of support • why do you have to do it, and what if you are infected? why? why does it have to be you? • when the patients' condition was bad and when we were having a hard time, no one showed appreciation of our hard work. identifying the best way to care for patients • after spending many days in the isolated room, we started using a messenger. we supported each other and shared information. it was very helpful for me to ask my colleagues when i was unsure about patient care. • we made a package for mers patients, a package for mers. at first, we brought water bottles to patients because they cannot come out a negative pressure room and we complained regarding this matter. next thing, we agreed to make a package for the patients in the isolation room. when a patient comes, we give this package that has water, sleeper, and disposable products that patients need. (continues) interviews to help understand the interviews. there were no repeat interviews carried out. the study was approved by the institutional review board (1041078-201506-hrsb-099-01). all participants were informed about the purpose of this study and participants' right to withdraw from the study at any time, without penalty. confidentiality of participants was ensured, and written informed consent was obtained from each participant. the responses from the participants were analysed, using qualitative content analysis (krueger & casey, 2009 ). data collection was conducted concurrently with data analysis and continued until no new information emerged from the responses. the researchers read each verbatim transcript several times to obtain an overall understanding of the content and to gain a sense of the whole. the meaning units (words, sentence, and paragraphs) in the interviews related to nurses' work experiences were identified and coded. the codes were sorted into similar things together and grouped into categories based on similarities and differences. after assessing themes across groups, overarching themes were derived. two of the investigators independently coded each transcript. when discrepancies in coding occurred, the investigators discussed and resolved them by consensus. trustworthiness of the study was maintained following criteria by lincoln and guba (1985) . the study participants included 25 female and 2 male nurses. their mean age was 29.5 (4.7) years, ranging from 20 to 24 years, and their work experience ranged from 3 months to 17 years. the following 4 major themes emerged: "experiencing burnout owing to the heavy workload," "relying on personal protective equip participants reported discomfort in wearing ppe all day on duty. the amount of time of wearing ppe varied according to their work and the severity of the patients' condition. participants said that they preferred a mask that led to less breathing difficulties. one participant said, "i prefer the mask made by a company because it has a space that helps me to breathe easily. many nurses prefer to use it." the patterns of staying in the isolation room wearing a papr differed. nurses from one hospital stated that they stayed in the isolation room for a maximum of 2 hours while wearing their papr and then came out; they stayed in the anteroom (a room in front of the negative pressure isolation room) and went back into the isolation room when needed. contrary to this, nurses from another hospital stayed in the isolation room for their entire shift, except for the lunch hour. meanwhile, nurses who wore a papr said that they felt like wearing a space suit. they had a backache from wearing heavy equipment. "i had put the battery of the papr on a side participants communicated with others by writing on paper. they reported that it was easy to hear the intercom sound in the room, but it was difficult to talk back because the head shield of the papr blocked out sounds. permission to use computers or smartphones in the isolation room varied across hospitals. in one hospital, nurses working in an isolated intensive unit communicated with other nurses in another isolated room using a smartphone messenger application. they said it helped them to know how others in isolation rooms were doing and to ask them when they were unsure about patient care. participants said that they gradually returned to normal life. the hospitals rewarded working with mers patients differently. these included participating in healing programs, receiving financial incentives, eating out in teams, or receiving several days off for resting. a participant said "i enjoyed participating in the healing camp. the post-trauma prevention education was also helpful. i was relieved to hear that we could seek psychiatric counselling if necessary." after completing their volunteered job with the mers patients, participants stated that they had learned on site while caring for infected patients and that it was very rewarding and worthwhile. they expressed that they felt being matured and gained a lot of confidence from these experiences. participants also said that when they returned to their work unit, they often heard "you did a good job" from their peers, and that "it felt supportive and healing." this study explored the nurses' work experience during the mers outbreak. our participants reported that their workload increased with time. this result indicates that, as part of emergency planning, nurse managers and hospital administrators should prepare for the extra workload during the emergency of an infection outbreak and to ensure quality of care. participants reported that restricting unauthorized access of visitors was one of the main issues. restricting visitors was one of the strategies used for controlling further outbreak during the norovirus outbreak (danial et al., 2016) . visiting hospitalized patients in a group is a part of the korean culture, as it is a way of expressing support and wishing for a quick recovery. rather than just restricting the visitors, it would be helpful to suggest alternative ways of expressing support for patients, such as sending a message through a phone or social networking service. participants expressed their concerns about the possibility of being infected. in fact, health care personnel who had close contact with mers patients were at a high risk for infection (alraddadi et al., 2016) . previous studies support our results. during the mers epidemic, health care workers felt fearful about being infected; however, they continued to work during the epidemic as it was their professional and ethical duty (al-dorzi et al., 2016; khalid, khalid, qabajah, barnard, & qushmaq, 2016) . emergency room nurses working during the outbreak of mers also expressed high concerns about being infected, and that they would have like to avoid caring for patients with mers if there was a choice (choi & kim, 2016) . these fears of nurses could be reduced by sharing the correct information about the quality of the protection devices they wear and appropriate ways to use them to prevent the transmission of infection (speroni et al., 2015) . in addition, hospitals experiencing mers epidemic suggested that institutional plans be made in advance to provide personal safety equipment when there is a rapid increase in its demand (al-dorzi et al., 2016; stirling, hatcher, & harmston, 2017) . however, our participants mentioned discomfort in wearing ppe. likewise, a study on a simulation exercise for health care workers wearing ppe in a hospital in the uk reported that they found the ppe uncomfortable, and even basic tasks took longer than usual while wearing it (phin et al., 2009) . thus, feedback from nurses on protection devices would help medical equipment companies design more comfortable medical protection equipment. our participants complained of having to continuously catch up with the frequently updated guidelines. likewise, it was reported that one of challenges for hospitals was the changing and conflicting guidelines and the overwhelming amount of information that required sifting through during the h1n1 influenza pandemic (rebmann, 2010 regarding going back to routines, our participants felt supported when they received positive responses from peers when they went back to their unit after taking care of patients at risk. additionally, hospitals implemented various programs for health care professionals to reward or appreciate their hard work during the outbreak. the common response of participants on these was very positive. in a study, nurses who took care of h1n1 high-risk infected patients and who worked in an isolated area in taiwan said that nurses needed counselling services (honey & wang, 2013) . after any outbreak, it may be important to offer a healing program for nurses, to help them share their experiences and feelings with others. a limitation of this study was that all participants were staff nurses. further research is needed to explore the mers experiences of patients, nurse managers, and other health care workers. because of variations in nurses' work schedule, the focus groups were quite small. the disadvantage of a small group is that it limits generating a rich diversity in views and the total range of experiences, although the advantage of smaller groups is that they are easier to recruit and allow everyone to have a greater opportunity to share experiences. another limitation was that this was a cross-sectional study. longitudinal studies are needed to examine the impact of any changes in hospital regulations or policies on nursing care. these study results suggest that nurse managers and administrative personnel should understand that overload of nurses' work during the outbreak may lead them burned out, which may negatively affect quality of care to patients. furthermore, establishing consistent and solid practice guidelines and efficiently disseminating them and training health care workers to deliver them could lead to less confusion during an infection outbreak. it is important to acknowledge nurses' work as valuable and to create a supportive environment in workplace of nurses. these efforts will empower nurses to work as an expert and will positively influence the quality of care. finally, it is essential to raise awareness about infection control among health care workers and people in general to strengthen emergency preparedness. the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study risk factors for middle east respiratory syndrome coronavirus infection among healthcare 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analysis communicating the changing role of a nurse in an epidemic: the example of the mers-cov outbreak in saudi arabia consolidated criteria for reporting qualitative research (coreq): a 32-item checklist for interviews and focus groups modeling the transmission of middle east respirator syndrome corona virus in the republic of korea middle east respiratory syndrome in 3 persons, south korea working experiences of nurses during the middle east respiratory syndrome outbreak none. the authors declare no conflict of interest. all listed authors meet the authorship criteria and all authors are in agreement with the content of the manuscript. sun-mi chae http://orcid.org/0000-0002-3010-2265 key: cord-283586-o8m6xdra authors: spanakis, nikolaos; tsiodras, sotirios; haagmans, bart l.; raj, v. stalin; pontikis, kostantinos; koutsoukou, antonia; koulouris, nikolaos g.; osterhaus, albert d.m.e.; koopmans, marion p.g.; tsakris, athanassios title: virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen date: 2014-12-31 journal: international journal of antimicrobial agents doi: 10.1016/j.ijantimicag.2014.07.026 sha: doc_id: 283586 cord_uid: o8m6xdra abstract serological, molecular and phylogenetic analyses of a recently imported case of middle east respiratory syndrome coronavirus (mers-cov) in greece are reported. although mers-cov remained detectable in the respiratory tract secretions of the patient until the fourth week of illness, viraemia was last detected 2 days after initiation of triple combination therapy with pegylated interferon, ribavirin and lopinavir/ritonavir, administered from day 13 of illness. phylogenetic analysis of the virus showed close similarity with other human mers-covs from the recent jeddah outbreak in saudi arabia. immunoglobulin g (igg) titres peaked 3 weeks after the onset of illness, whilst igm levels remained constantly elevated during the follow-up period (second to fifth week of illness). serological testing confirmed by virus neutralisation assay detected an additional case that was a close contact of the patient. an upsurge of middle east respiratory syndrome coronavirus (mers-cov) infection has been recently described in countries of the arabian peninsula resulting in exported cases from these countries to the european union [1] . cases of mers-cov infection are associated with a high case fatality rate since there is no available treatment. there is a scarcity of data on specific therapeutic interventions for the disease. published reports propose the use of known antivirals based on extrapolation of data from: (i) the severe acute respiratory syndrome (sars) epidemic that was also associated with the circulation of a novel coronavirus; (ii) in vitro data; (iii) animal experimental infections and therapy data; and (iv) limited clinical data for actual mers-cov infections [2] [3] [4] . however, no clear-cut recommended therapeutic regimen exists and the evidence for grading such interventions is generally low, with the exception of the use of convalescent serum that based on biological effects is given the highest grade [5] . moreover, little is known about the viral kinetics of mers-cov-associated infection, especially when a specific antiviral or other therapeutic intervention is attempted. a case of mers-cov has recently been described in greece in a traveller who had extensive contact with the healthcare environment in jeddah (saudi arabia) [6] . here we describe molecular, serological and phylogenetic analyses of this case as well as evidence for a second case that was a close contact of the first patient. furthermore, we provide evidence of the kinetics and the pattern of viral excretion in biological specimens obtained from the first greek case while the patient was on a triple antiviral regimen. a preliminary report of the first imported, laboratory-confirmed mers-cov case in greece has been described elsewhere [6] . a full description of the course of illness and treatment regimen in http relation to kinetics of virus shedding and immune response was prepared by review of the patient records. in the course of the outbreak investigation, 40 of 75 patient's contacts, including the patient's wife, provided an oropharyngeal sample for pcr testing 1 week after contact with the positive case; 5 additional contacts were included in the serology examination group. all were submitted to personal clinical monitoring for fever and upper respiratory infection symptoms and were advised to call the hellenic centre for disease control and prevention (cdc) command centre immediately in such an instance. in addition, all were offered the chance to provide serum samples on a voluntary basis for specific anti-mers antibody testing at baseline (same time as the oropharyngeal pcr testing) and 3 weeks after exposure. during the patient's stay in the intensive care unit (icu), samples from the oropharynx, trachea, urine and faeces were tested for diagnostic evaluation and to monitor viral shedding. a real-time reverse transcription pcr (rt-pcr) method based on amplification of the upstream envelope gene (upe), the nucleocapsid (n) gene and the open reading frame (orf) 1a of the virus was used for detection of mers-cov according to previously described methodology [7, 8] . immunoglobulin g (igg) and igm antibody titres in serum samples were determined using an anti-mers-cov indirect immunofluorescence assay (euroimmun ag, lübeck, germany). confirmation of the serological findings was performed with a virus neutralisation assay as described previously [9] . samples from the patient's upper respiratory tract underwent conventional or molecular testing for the presence of other respiratory pathogens: thus, cultures applied for bacterial testing, whilst real-time rt-pcr was performed for several respiratory viruses including influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza, adenovirus, enterovirus, bocavirus and human metapneumovirus (hmpv) (m.w.s. r-gene; biomérieux, marcy-l'étoile, france). specific urine antigen testing of urine samples was utilised for legionella pneumophila and streptococcus pneumoniae (binaxnow ® ; alere, orlando, fl). a stool culture was performed due to a history of possible typhoid fever, diagnosed by treating physicians in saudi arabia [6] . nucleotide sequences of 3-kb concatenated sequences of representative mers-covs were analysed and a phylogenetic tree was constructed by the phyml method as described previously [10] . a 69-year-old patient of greek origin who was a permanent resident of jeddah presented to a tertiary care centre a few hours after arriving in athens (greece) on 17 april 2014. his chief complaints included fever since 8 april 2014 and diarrhoea since 10 april 2014. the most likely source of exposure was the hospital environment in jeddah. the patient had no known co-morbidities. at the time of initial evaluation, a fever of 38.3 • c was noted together with low oxygen saturation (92%), although the patient exhibited minimal respiratory symptoms. a chest radiograph depicted bilateral lung infiltrates consistent with viral pneumonia. the patient was immediately placed under isolation because of suspicion of mers-cov infection, and an antimicrobial regimen targeting communityacquired pneumonia was initiated. on 18 april 2014, mers-cov infection was confirmed by means of viral rna detection in a pharyngeal swab at the department of microbiology, university of athens medical school (athens, greece). after laboratory confirmation of mers-cov, the patient was transferred to a specialised respiratory disease unit in the 'sotiria' chest diseases hospital of athens where he was treated in a negative pressure regular room in isolation until 20 april 2014 when, due to deterioration of his respiratory function and development of acute respiratory disease syndrome (ards), he was intubated, ventilated and transferred to a negative pressure room in the icu of the same hospital. an empirical antiviral regimen was initiated on day 13 of illness consisting of oral (p.o.) lopinavir/ritonavir (400/100 mg twice daily), pegylated interferon (180 g subcutaneously once per week for 12 days) and ribavirin (2000 mg p.o. loading dose, followed by 1200 mg p.o. every 8 h for 8 days) based on available evidence [3] [4] [5] 11, 12] (fig. 1) . the patient remained intubated exhibiting hypoxia and occasionally hypercapnia while breathing inspired oxygen in the range of 0.45-0.60. he remained febrile with a plateau temperature of >39 • c and a maximum value of 40.5 • c on day 18 of illness. fever started subsiding below 38 • c on day 22. acute kidney injury was diagnosed on day 16 of illness and rapidly progressed to non-oliguric renal failure that reverted to rifle injury level (i.e. two-fold increase in the serum creatinine, or glomerular filtration rate decrease by 50%, or urine output <0.5 ml/kg/h for 12 h) on day 21. the patient's diarrhoea resolved gradually starting on day 13 and he developed constipation thereafter with normalisation of his bowel movements and gastrointestinal function on day 19. owing to development of jaundice and hyperbilirubinaemia attributed to ribavirin [13] , the drug was discontinued on day 20. during the course of his hospitalisation, the patient was diagnosed with adenocarcinoma of the colon and eventually died from septic shock 2 months and 19 days after the initial diagnosis. cultures and antigen detection were negative for l. pneumophila and s. pneumoniae. virological testing was negative for the presence of any other respiratory virus. no relevant enteric pathogens were identified as a cause of the patient's diarrhoea. rna was detected in several consecutive patient samples from different sites that included faecal material and serum (fig. 1) . shedding of mers-cov in the respiratory secretions of the patient was noted until the fourth week of illness, whereas viraemia was last detected 15 days after onset of illness and 2 days after initiation of the triple combination antiviral regimen. consecutive urine testing did not reveal the presence of mers-cov rna (fig. 1) . serological testing showed a peak igg titre during the third week of illness, whilst during the fourth and fifth weeks igg titres were substantially declining. igm titres were persistently elevated during the whole survey period (day 13 until day 34 of illness) (fig. 1 ). viral neutralisation assays performed at erasmus medical center (rotterdam, the netherlands) confirmed the immunofluorescence testing results. initial and follow-up serological testing were performed on serum samples from 45 patient's contacts. seroconversion was revealed in one of them who developed an igg titre of 1/500 and an igm titre of 1/100 at 21 days after making contact with the patient. this was a 63-year-old man with a past medical history of coronary artery heart disease and diabetes. the presence of specific mers-cov antibodies was confirmed by the virus neutralisation assay. without other symptoms from any other system. no nasopharyngeal pcr testing was performed at the time since he was outside the incubation period of 14 days. he has been well since then and during the time that he was symptomatic he only had contact with his family members (four persons). the initial patient's wife had a brief episode of fever on 30 may 2014. oropharyngeal pcr testing was negative for mers-cov, and all contacts remained seronegative on repeat testing. partial genomic sequencing [14] revealed the close phylogenetic relationship with clinical mers-cov strains associated with severe respiratory infection from patients in jeddah (fig. 2) . in this report, we further characterised serological and virological parameters of the first mers-cov case in greece. rising titres of igg were demonstrated in sequential serum samples, with the peak titres approximately 3 weeks into the course of the disease. this is in accordance with serological testing guidance from the world health organization (who) recommending baseline testing from initial contact with an affected case and repeated serological testing on day 21 [15] . on the other hand, igm titres of the patient remained constantly elevated above the threshold of detection, albeit at a lower level than igg antibodies, for a prolonged period of ≥1 month of follow-up. thus, isolated use of igm testing without concomitant igg determination appears not to be sufficient to reveal a recent infection. it should be noted, however, that in the absence of detailed studies, use of serological testing for mers-cov detection in humans needs to be further evaluated. prolonged shedding of the virus was noted from the respiratory tract of the patient. this finding is consistent with data regarding the sars coronavirus. in a report dealing with patients affected by sars, prolonged shedding of the virus was noted in stool (up to 126 days) and respiratory specimens (up to 52 days) [16] . data regarding the length of mers-cov excretion from different body sites are scarce [17] . excretion of the virus probably depends on the amplitude of replication in different body sites, the underlying immune status and co-morbidities, and appropriate antiviral therapy. the non-detectable viral rna in serum by day 3 after initiation of the antiviral treatment could be explained either by viral clearance in an otherwise immunocompetent person or by effectiveness of the instituted antiviral regimen. literature on appropriate antiviral intervention for mers-cov is very limited and currently no evidence-based therapy exists. the regimen chosen was based on the best available literature as well as evidence from animal and patient data that have been described elsewhere [2] [3] [4] . the role of interferon therapy for mers-cov infection needs to be further elucidated. an attenuated interferon-␤ (ifn-␤) response has been described as a result of mers-cov infection [18] and extensive use of interferon-based regimens alone or in combination with ribavirin has been described for sars [4] . however, interferons appear to have a better antiviral effect on mers-cov compared with sars-cov in in vitro experiments [19] . in vitro, ifn-␤ appears to exhibit the best anti-mers-cov effect [20] . interferon activity has been enhanced by the addition of ribavirin in in vitro experiments [21] . furthermore, this combination has shown promising clinical and radiological effects in rhesus macaques experimentally infected with mers-cov [12] . thus, the clinical team elected to use this combination despite the fact that a prestigious public health agency ranks ribavirin use as not supported by high-quality evidence [5] . in a more recent update published by the same public health agency, the use of interferons and lopinavir is ranked under the recommendation of benefit is likely to exceed risk, whereas the combination of interferon and ribavirin is ranked as data are inadequate for assessment [22] . the frequent side effects of ribavirin limit its use in combination regimens for actual mers-cov-infected patients, as was the experience with the current patient where liver toxicity, although not definitively associated, was mainly attributed to this medication. the renal function deterioration of the patient described here was considered multifactorial and probably also a complication of the virus infection [23] . the possibility that drug toxicity might have contributed in the renal dysfunction could not be excluded, however. no drug levels were measured since the patient was under continuous renal replacement therapy at that time and drug levels would be unreliable. in the actual clinical human setting, the combination of ribavirin and interferon has been tried, with no successful outcome reported among any of the mers-cov-infected recipients [3] . nevertheless, the group studied consisted of severely ill patients who received this combination quite late in the course of their disease [3] . lastly, we added the protease inhibitors lopinavir/ritonavir based on experience accumulated from the sars epidemic where the addition of this agent to ribavirin improved the outcome of infection [24] . expanding the knowledge regarding viral kinetics and the pattern of shedding especially in association with specific therapeutic interventions has important implications for infection control in the healthcare environment, especially as it relates to potential transmission to other patients and healthcare workers [25] . phylogenetic analysis of the greek mers-cov strain showed close similarity with circulating patient viral strains from the recent jeddah outbreak as well as with a strain isolated from a dromedary camel in qatar. this is in accordance with previous genetic studies that have shown identical viral strains between infected humans and dromedary camels and generated the hypothesis that dromedary camels are among the reservoirs of the virus in nature [10, 26] . also, the presence of mers-cov-specific antibodies in camels across a wide geographic area in africa and the arabian peninsula signifies the possibility for zoonotic transmission between camels and humans [27] [28] [29] . the potential for transmission across different individual strains should be further explored. in this patient, the most likely source of exposure was the hospital environment in an endemic area, as his wife was hospitalised in a local hospital in jeddah [6] . it appears that healthcare-associated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic in the recent upsurge [30] . in conclusion, we describe the genetic stability of the mers-cov in a strain from the recent jeddah outbreak. although reassuring, this finding should not limit the level of awareness regarding the increased number of cases in the arabian peninsula reported recently and the potential evolution and more efficient transmission of the virus. a who committee recently concluded that the conditions for a public health emergency of international concern have not yet been met. nevertheless, important gaps in current knowledge about mers-cov exist. more investigations to clarify the natural reservoir and modes of transmission are necessary. persistence of virus shedding in patients' secretions and the effect of immune status and antiviral therapy together with the implementation of appropriate infection control measures are of paramount importance in limiting further spread of this potentially lethal virus. funding: this work was funded by a grant from the netherlands organisation for scientific research (nwo) [no. 40-00812-98-13066]. competing interests: none declared. ethical approval: not required. european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov). ecdc what are 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coronavirus (mers-cov): a preliminary report of two cases treatment with interferon-␣2b and ribavirin improves outcome in mers-covinfected rhesus macaques schering corp product information. rebetol® (ribavirin, usp). kenilworth, nj: schering corp isolation of mers coronavirus from dromedary camel seroepidemiological investigation of contacts of middle east respiratory syndrome coronavirus (mers-cov) patients. geneva: switzerland: who long-term sars coronavirus excretion from patient cohort clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential interferon-␤ and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays inhibition of novel ␤ coronavirus replication by a combination of interferon-␣2b and ribavirin in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 the authors would like to acknowledge the department of epidemiological surveillance and response of the hellenic centre for disease control and prevention (cdc), and especially dr georgia spala, theano georgakopoulou and agoritsa baka, for mers-related activities, as well as dr spyros sapounas for contacting the case investigation of the second case. key: cord-311937-6hadssmh authors: sherbini, nahid; iskandrani, ayman; kharaba, ayman; khalid, ghalilah; abduljawad, mohammed; al-jahdali, hamdan title: middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data date: 2016-06-11 journal: j epidemiol glob health doi: 10.1016/j.jegh.2016.05.002 sha: doc_id: 311937 cord_uid: 6hadssmh background: middle east respiratory syndrome coronavirus (mers-cov), is an emerging virus respiratory infection. it has a high mortality rate and a wide spectrum of clinical features. this study describes the clinical characteristics and outcome of mers infected patients. methods: a retrospective study was conducted of all confirmed mers-cov infections from march 2014 to may 2014 at two tertiary care hospitals in al-madinah region (saudi arabia). we gathered data about demographic, clinical presentation, and factors associated with severity and mortality. results: a total of 29 cases were identified; 20 males (69%) and nine females (31%), age 45 ± 12 years. the death rate was higher for men (52%) than for women (23%). initial presentation was fever in 22 (75%) cases, cough in 20 (69%) cases, and shortness of breath in 20 (69%) cases. associated comorbidities were diabetes mellitus in nine (31%) patients and chronic kidney disease (ckd) in eight (27%) patients. duration of symptoms before hospitalization ranged from 2.9 days to 5 days. elevated liver enzymes were present in 14 (50%) patients and impaired renal profile present in eight (27%) patients. we also describe in this study radiological patterns and factors associated with mortality. conclusion: mers-cov infection transmission continues to occur as clusters in healthcare facilities. the frequency of cases and deaths is higher among men than women and among patients with comorbidities. in saudi arabia, a beta new coronavirus was isolated for the first time at the end of 2012 from a patient who presented with acute community acquired pneumonia [1] . he died 11 days later from progressive severe respiratory failure and acute renal failure (arf) and his sputum sample was negative for respiratory viruses commonly tested. epidemiology of middle east respiratory syndrome coronavirus (mers-cov) was expanded after exploring the large hospital outbreak in al-hasa, saudi arabia [2] . subsequently, the virus was designated as mers-cov [3] . the geographic distribution of the cases has been mainly linked to the arabian peninsula particularly from saudi arabia where most of the cases were reported [4] [5] [6] [7] . however, in some countries in north america, europe, africa, and asia, the disease has been detected in some travelers from endemic countries [3, [7] [8] [9] [10] [11] [12] [13] . the initial occurrence of mers-cov was thought to have particular predominance for male patients and those with comorbid diseases. the male-tofemale ratio was between 2.8:1 and 3.3:1 [2, 6] ; this male predominance might have been related to the nature of the outbreak. initial cases were reported among elderly patients with a median age of 56 years. mers-cov has a very high mortality rate, and complications arising from infection can result in severe respiratory and renal failure [2] . symptoms of mers-cov range from mild upper respiratory symptoms to rapidly progressive severe pneumonia requiring intubation, and multiorgan failure. a significant number of patients may present with nonrespiratory symptoms such as headache, myalgia, and gastrointestinal symptoms of nausea, diarrhea, or vomiting [2, 14] . this study describes the demographic, clinical characteristics, and outcome of mers-cov in al-madinah region, saudi arabia. a retrospective chart review study of all confirmed mers-cov cases recorded by two tertiary hospitals from the madinah region from march 2014 to may 2014. institutional review board approval was obtained for our study from authorities of both hospitals. a case was confirmed as having infection if mers-cov real-time polymerase chain reaction was positive, using the recommended sampling technique (nasopharyngeal swab and tracheal aspirates or bronchoalveolar lavage in intubated patients). extraction of rna was performed with roche magna pure lc (rna viral isolation kit). samples were pretreated with lysis according to the manufacturer's instructions [15] . we obtained data about demographic characteristics, clinical presentation, laboratory results, diagnosis, incubation period, smoking history, comorbidities, and history of contact with camels or mers-cov positive patients in regions within the madinah area. we recorded the duration of the patient's illness, microbiological test results, and reviewed imaging and treatments received. we also recorded the following outcomes: duration of mechanical ventilation, intensive care unit (icu) length of stay, and survival during hospitalization until the patient is discharged from hospital. data were analyzed using ibm spss for windows, version 18.0. the frequency of cases of mers-cov infection and percentage of resulting deaths were calculated. statistical analyses of demographics, clinical, and laboratory descriptive data are tabulated. descriptive statistics such as means and standard deviation mean (±sd) were used to describe the age of the patients, laboratory test results, and duration of illness. frequencies and percentages n (%) were used to describe demographic and mer-cov outcomes. we also did a correlation with the outcome using the t test and fisher's exact test as appropriate with a significant value at p 6 0.05. the total number of cases with confirmed mers-cov infection reported from april 2014 to may 2014 was 29. the majority of patients (60%) were men, and the median age was 45 years. the most common symptoms were fever (75.9%) and cough (69%), shortness of breath (69%), and vomiting and diarrhea (27%). the average duration of symptoms prior to hospitalization was 5 days (range, 1-12 days). demographic and clinical characteristics of patients with confirmed mers-cov are shown in table 1 . mortality rate among patients with confirmed mer-cov was 34%. mortality from mers-cov was significant (p < 0.05) and associated with older age, the presence of gastrointestinal symptoms, longer duration of symptoms prior to hospitalization (8 ± 2.5 days), diabetes mellitus, chronic kidney disease (ckd), smokers, and lower blood pressure, as shown in table 1 . most of the patients were coming from hanakia 11 (38%) and all 11 patients had contact with camels table 2 . close contact with confirmed index mers-cov was documented in five patients, all of whom were healthcare professionals; three staff nurses and two clinicians. most of the patients were mildly hypoxic; oxygen saturation was 88.9 ± 5.4 at presentation with a picture of respiratory acidosis (ph 7.3 ± 0.1 and pco2 57.1 ± 8.2). the basic liver functions show elevated alanine transaminase (98.4 ± 105.4 u/l), aspartate aminotransferase (86.3 ± 93.5 u/l), and creatinine (225.0 ± 115.3 mmol/l). as shown in fig. 1 , which were collected during the patient's initial assessment. all patients had abnormal initial chest radiographs. the predominant finding was bilateral basal consolidations with ground-glass opacities, nodular or/and reticular pattern, and total diffuse multilobar involvement. all patients had appropriate supportive management and received a broad spectrum antibiotic and readjusted based on sputum cultures. among patients who required icu, the mean time of icu stay ranged from 9 days to 55 days (13.7 ± 4.0 days), and mechanical ventilation support was used in nine (31%) patients. mechanical ventilation support and longer stay in icu were significantly associated with death (p < 0.05) (see table 4 ). emerging viral respiratory infections are causing a significant burden on public health and causing significant morbidity and mortality. over the past decades, many viral infection outbreaks have been reported including influenza h7n9 such as h1n1, sars-cov, and the most recent mers-cov infection. the world health organization reported 1368 laboratory-confirmed cases of human infection with mers-cov including at least 487 deaths between 2012 and july 2015 [7] . they reported that 65% of cases were male (n = 1359) and the median age was 50 years (n = 1365) which is similar to our study. similar to a study reported by assiri et al. [2] , our study showed more cases among older patients, but our study showed an association of death with older age. since 2012, 26 countries have been affected, including countries in the middle east, africa, europe, asia, and north america as reported from the world health organization. the majority of cases (75%) have been reported from saudi arabia. in saudi arabia, mortality secondary to mers-cov was 35% [16] and in our study it was 37% (al-madinah). camels have been confirmed as a reservoir for mers-cov, and many hypotheses are behind this zoonotic (camels) transmission [17, 18] . in our study, only five healthcare employees acquired infection from documented contact with an infected patient, but another 24 patients were coming from areas around al-madinah; the largest number of infected patients was from the alhenakia area, where camels are prevalent. the second important mode of transmission is person-toperson transmission (travelers returning from the middle east and close contacts with mers-cov cases) [19] . this type of transmission was confirmed by genome sequencing of mers-cov [19] and isolates from the al-hasa healthcare-associated outbreak [16] . in the uk, mers-cov was transmitted to a family member who visited a patient with confirmed infection and another report from france described patient-to-patient nosocomial transmission of mers-cov. in our study, five (18%) patients had transmission through close contact. approximately 30% of mers-cov patients in our study reported diabetes and ckd, which is similar to those from other observational epidemiological studies in saudi arabia [2, 16] . the high mortalities reported early were probably due to a delay in the diagnosis and presence of comorbidities [20] . however, the large number of mers-cov cases and ckd might have been based on the al-hasa hospital outbreak, which mainly happened in the dialysis center [2] . we may consider the existence of chronic illnesses such as diabetes, hypertension, and ckd increasing the risk of acquiring this infection and categorize them as a high risk group for more complications and worse outcome as was revealed [21] also in our study. we and others have found that the severity of illness associated with mers-cov infection ranges from mild to fulminant [22] . the severity of the respiratory infections caused by mers-cov can progress to hypoxemic respiratory failure which requires the use of mechanical ventilation and death [23] . all of our patients had significant respiratory manifestations requiring admission to the icu but 30% only required mechanical ventilation and died [24] . mers-cov is known to infect cell lines of the intestinal tract [25] , but it is not yet known what proportion of ill patients shed the virus in their stools, which is why some patients presented with gastrointestinal symptoms. identification of the full range of clinical presentations is important so that the mild cases are not missed. mers-cov is detected by reverse transcription polymerase chain reaction. to date, laboratory testing for mers-cov remains not very accurate; the sensitivity and negative predictive values are unknown. development of rapid and accurate diagnostic tests is needed urgently. results of throat swabs were occasionally negative and repeat testing for mers-cov is recommended. it seems difficult to conclude that one negative sample is enough to rule out mers-cov disease when a patient presents with respiratory symptoms and history of exposure. it is also not clear whether nasopharyngeal samples might be superior to throat samples or whether virus is shed more abundantly later in the course of the illness as it is in sars. there is evidence that repeat testing and tests on sputum or bronchoalveolar lavage fluid are of value in improving diagnostic accuracy. microbiological investigations were done routinely to exclude bacterial copathogens with community acquired pneumonia (cap). we had seven patients with methicillin-resistant staphylococcus aureus (mrsa) coinfection, two with streptococcus and one with methicillin-sensitive staphylococcus aureus (mssa) in our study population. assiri et al. [2] stated that none of the 47 samples screened was positive. other investigators found that one patient was coinfected with mssa and influenza b and another with streptococcus pneumonia [23] . there might have been a selection bias because we were only screening critically ill cases, which in turn will lead to detection of more severe cases of mers-cov infection; mild cases may not come to hospital or may not be screened for mers-cov and could lead to false high case-fatality rates. clinical symptoms, laboratory investigations, and imaging findings of mers-cov are similar to those noted in other community-acquired respiratory tract infections. radiological findings in mers-patients tended to range from unilateral focal air-space opacities to multifocal or bilateral lower lobe involvement was seen with a picture of organizing pneumonia which was noted in our patients and other reports [25, 26] . on the basis of findings until now, the clinical features of mers-cov infection have similarities to those seen in patients with sars-cov infection. the initial phase of nonspecific fever, cough, and shortness of breath are the major symptoms in those admitted to hospital; other common symptoms include chills, rigor, headache, myalgia, and malaise which may last for several days before progressing to pneumonia [21, 22] . a significant number of patients had gi symptoms, another important similarity to sars. we found patients with mers-cov who had gi manifestation tend to progress to severe illness and this may be considered one of the poor prognostic factors. the disease may progress rapidly to a critical respiratory failure, requiring mechanical ventilation and lead to death in the icu [5] . in our observations, all of the patients started with symptoms of fever, cough for 5-7 days. our study design has several limitations including that it is a retrospective chart review study with known inherited problems; these include missing data regarding contact with camels and documentation of all comorbidities and availability of follow up data after discharge. despite these limitations, we have been able to highlight some features in the epidemiological, demographic, and clinical characteristics of patients with mers-cov infection in al-madinah regions. the epidemiology and the transmission pattern of mers-cov to date indicate that the majority of cases occur in the healthcare setting. strengthening the infection control measures in the healthcare setting is of great importance. since about 25% of cases are community based, there is a real need to further prevent the animal-to-human transmission of mers-cov. the frequency of cases and deaths is higher among men than women and those around 45 years of age are the most affected patients. the disease had higher mortality in older patients with comorbidities. also, the presence of gastrointestinal manifestations, high liver enzymes, and need for mechanical ventilation or longer stay in icu are all associated with high mortality. there are gaps in our knowledge of the epidemiology, prevalence, clinical characteristics, prognostic factors, and nature of the disease. it is also important to further delineate the transmission routes and the presence of any other animal or intermediate hosts. the influence of geographical distribution and comorbidities on the incidence and outcome of mers-cov patients should be studied further. the authors report no conflicts of interest in this work. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group managing mers-cov in the healthcare setting epidemiological and clinical aspects of coronavirus infection mers-cov middle east respiratory syndrome novel corona mers-cov infection. epidemiology and outcome update middle east respiratory syndrome coronavirus (mers-cov), summary of current situation, literature update and risk assessment. who/mers/ra/151. geneva: who lessons to learn from mers-cov outbreak in south korea epidemiological investigation of mers-cov spread in a single hospital in south korea public health response to two incidents of confirmed mers-cov cases travelling on flights through london heathrow airport in 2014. lessons learnt travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia eds on heightened alert for mers-cov as first cases reach the us middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study hospital outbreak of middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012 family cluster of middle east respiratory syndrome coronavirus infections prevalence of diabetes mellitus in a saudi community severe acute respiratory syndrome and coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection a major outbreak of severe acute respiratory syndrome in hong kong clinical and laboratory features in the early stage of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings this study did not receive funding. key: cord-286631-3fmg3scx authors: pormohammad, ali; ghorbani, saied; khatami, alireza; farzi, rana; baradaran, behzad; turner, diana l.; turner, raymond j.; bahr, nathan c.; idrovo, juan‐pablo title: comparison of confirmed covid‐19 with sars and mers cases ‐ clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta‐analysis date: 2020-06-05 journal: rev med virol doi: 10.1002/rmv.2112 sha: doc_id: 286631 cord_uid: 3fmg3scx introduction: within this large‐scale study, we compared clinical symptoms, laboratory findings, radiographic signs, and outcomes of covid‐19, sars, and mers to find unique features. method: we searched all relevant literature published up to february 28, 2020. depending on the heterogeneity test, we used either random or fixed‐effect models to analyze the appropriateness of the pooled results. study has been registered in the prospero database (id 176106). result: overall 114 articles included in this study; 52 251 covid‐19 confirmed patients (20 studies), 10 037 sars (51 studies), and 8139 mers patients (43 studies) were included. the most common symptom was fever; covid‐19 (85.6%, p < .001), sars (96%, p < .001), and mers (74%, p < .001), respectively. analysis showed that 84% of covid‐19 patients, 86% of sars patients, and 74.7% of mers patients had an abnormal chest x‐ray. the mortality rate in covid‐19 (5.6%, p < .001) was lower than sars (13%, p < .001) and mers (35%, p < .001) between all confirmed patients. conclusions: at the time of submission, the mortality rate in covid‐19 confirmed cases is lower than in sars‐ and mers‐infected patients. clinical outcomes and findings would be biased by reporting only confirmed cases, and this should be considered when interpreting the data. during the last two decades, coronaviruses have been recognized as one of the most critical human pathogenic viruses that affect global health and cause concern in the world health system. 1 coronavirus is classified into four genera: alpha, beta, delta, and gamma. major (about 4% mortality). 8 there is no vaccine or targeted treatment currently available for covid-19 infection. treatment is mostly supportive, although multiple experimental antiviral medications are being evaluated. 9, 10 thus, prevention and rapid diagnosis of infected patients are crucial. the trigger for rapid screening and treatment of covid-19 patients is based on clinical symptoms, laboratory, and radiographic findings that are similar to sars and mers infections. in this study, we attempted to distinguish the clinical symptoms, laboratory findings, radiographic signs, and outcomes of confirmed covid-19, sars, and mers patients. all findings are compared to determine unique features among each of them. these data could be helpful in the early diagnosis and prevention of infection as well as providing more reliable epidemiological data on a large-scale for health care policies and future studies. this study was conducted according to the preferred reporting items for systematic reviews and meta-analyses statement (prisma) guidelines, and it has been registered in the prospero database (id 176106). 11 we searched all studies published up to 28 february 2020, from the following databases: embase, scopus, pubmed, web of science, and the cochrane library. search medical subject headings (mesh) terms used were: "covid-19," "coronavirus," "severe acute respiratory syndrome," "sars virus," "severe acute respiratory syndrome coronavirus 2", "coronavirus infections," "middle east respiratory syndrome coronavirus," and all their synonyms like "wuhan coronavirus," "sars-cov-2," and "covid-19," "2019-ncov" and mers. moreover, we searched for unpublished and grey literature with google scholar, centre for disease controls (cdc) and who databases. we also examined references of included articles to find additional relevant studies. there was no language restriction, and all included studies were written in english or chinese languages; the latter was translated by https://translate.google.com/. additional search strategy details are provided in table s1 . duplicate studies were removed using endnote x7 (thomson reuters, new york, ny, usa). records were initially screened by title and abstract by independently four authors (ap, sg, ak, and rf). the full-text of potentially eligible records was retrieved and examined, and any discrepancies were resolved by consensus. studies had to fulfill the following predetermined criteria to be eligible for inclusion in our meta-analysis. all case-control, cross-sectional, cohort studies, case reports, and case series peer-reviewed studies were included if they reported the number of confirmed cases of patients with demographic data, [and] [or] clinical data, [and] [or] laboratory data, [and] [or] risk factor data. studies were excluded if they did not report the number of confirmed cases. letters to the editor, individual case reports, review articles, and news reports were also excluded. duplicate information from the same patient was combined and counted as a single case when the data was reported twice. all covid-19 included publications were published in 2020, and all patients were from china. the following items were extracted from each article: first author, center and study location in china, countries, sample collection time, patient follow-up time, the reference standard for infection confirmation, number of confirmed cases, study type, and all demographic, clinical, laboratory data, and risk factor data. three of our authors (sg, ak, and rf) independently extracted data, and all extracted data were checked randomly by another author (ap); differences were resolved by consensus. quality assessments of studies were performed by two reviewers independently according to the critical appraisal checklist recommended by the joanna briggs institute, 12 and disagreements were resolved by consensus. the checklist is composed of nine questions that reviewers addressed for each study. the "yes" answer to each question received one point. thus, the final scores for each study could range from zero to nine (table s2 ). data cleaning and preparation were done in microsoft excel 2010 (microsoft©, redmond, wa, usa), and further analyses were carried out via comprehensive meta-analysis software version 2.0 (biostat, englewood, nj). determination of heterogeneity among the studies was undertaken using the chi-squared test (cochran's q) to assess the appropriateness of pooling data. depending on the heterogeneity test, we used either random or fixed-effect models for pooled results. in the case of high heterogeneity (i2 > 50%), a random effect model (m-h heterogeneity) was applied, while in low heterogeneity cases (i2 < 50%), a fixed-effect model was used. 13 percentages and means ± sds were calculated to describe the distributions of categorical and continuous variables, respectively. p values reflect study heterogeneity with <.05 being significant. we also used the funnel plot, begg's, and egger's tests based on the symmetry assumption to detect publication bias ( figure s1 ). the process of study selection is displayed in figure 1 quality assessment of included studies was performed based on the critical appraisal checklist, and the final quality scores of the included studies are represented in table s2 . in brief, studies by chen et al, 14 wang et al, 17 huang et al, 18 guan et al, 19 zhang et al, 24 cheng et al, 25 li et al, 28 xu et al, 30 figure s2 ). cough was the second most common symptom presenting in covid-19 63% (95% ci 55.5-70, p < .001), sars 54.2% (95% ci 49-59, p < .001), and mers 61% (95% ci 51-70, p < .001) of patients ( figure s3 ). age is an exception, presented in mean age in years. (95% ci 2.7-13, p < .001), or runny nose 6% (95% ci 1-14, p < .001). more detail information about demographics and clinical characterization of covid-19 (table s3) , sars (table s4) , and mers patients (table s5 ) demonstrated in the supplementary material. the greatest risk for covid-19 patients 69.5% (95% ci 54.5-81, p < .001) up to 28 february 2020, is a history of recent travel to wuhan, contact with people from wuhan, or were wuhan residents, and 24.3% (95% ci 9.6-49, p < .001) had exposure at the seafood market(s (table s9) , sars (table s10) , and mers patients (table s11) is demonstrated in the supplementary material. most covid-19 confirmed patients required hospitalization 85.4% (95% ci 68-94, p < .001) and 20.6% (95% ci 6.7-48, p < .001) were deemed to be in critical condition. the mortality rate of covid-19 confirmed cases was 5.6% (95% ci 2.5-12.5, p < .001), sars 13% (95% 9-17, p < .001), and mers 35% (95% ci 31-39, p < .001) (figure 2 ). the laboratory findings showed that among a subset of patients 4.5% increased the percentage value. the actual mortality rate from covid-19 is almost certainly much lower than that found in this study. as more data emerges from screening asymptomatic or mildly symptomatic individuals in china and around the world, the exact mortality rate will be better understood. among covid-19, sars, and mers patients, leukocytosis was found in 13.3%, 28%, and 30%, respectively, and leukopenia in 26%, 32%, and 41%, respectively. most of the patients with coronavirus had abnormal chest radiological findings. on the other hand, runny nose and rhinorrhea are less common symptoms in coronavirus-infected patients, 127 and that it is typically expressed on pulmonary alveolar epithelial cells. 128 another study reported that following covid-19 infection deregulated cytokine/chemokine response and higher virus titer causes an inflammatory cytokine storm with lung immunopathological injury. 129 inflammation related to the cytokine storm in the lungs may then spread throughout the body via the circulation system. covid-19 patients have been reported to have increased plasma concentration of inflammation-related cytokines, including interleukin (il)-2,6,7,10, tumor necrosis factor-α (tnf-α), and monocyte chemoattractant protein i (mcp-i) especially in moribund patients. 130 several limitations of this study exist. publication bias and study heterogeneity are unavoidable in this type of study. therefore, it should be considered when interpreting the outcomes of the reports and our final data set. furthermore, this study likely overestimates disease severity due to a lack of screening for asymptomatic or mildly symptomatic individuals and subsequent publication bias related to these factors. likely, many infected persons have not been detected, thus falsely elevating the rates of hospitalization, critical condition, and mortality. the lower quality analysis and reporting in some of the included publications is another limitation of the study. to prevent language bias, we included reports in languages other than english. additionally, we searched for a variety of sites and databases to prevent internet platform bias. using egger's regression test, we did not find significant publication bias. journal bias is an issue facing those who carry out a meta-analysis, yet it does not usually affect the general conclusions. 132 however, we cannot reject the occurrence of other biases in this study, such as choice bias, since several journals are not indexed in embase, scopus, pubmed, web of science, and the cochrane library and unpublished data from some regions of the world. fever and cough are the most common symptoms of covid-19-, sars-, and mers-infected patients. the mortality rate in covid-19 confirmed cases was lower than sars-and mers-infected patients. clinical outcomes and findings may be biased by reporting only confirmed cases, and it should be considered when interpreting the data. a novel 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to cite this article comparison of confirmed covid-19 with sars and mers cases -clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta-analysis none. the authors have declared that no conflict of interests. key: cord-307995-8q7efrqk authors: al-tawfiq, jaffar a.; omrani, ali s.; memish, ziad a. title: middle east respiratory syndrome coronavirus: current situation and travel-associated concerns date: 2016-05-04 journal: front med doi: 10.1007/s11684-016-0446-y sha: doc_id: 307995 cord_uid: 8q7efrqk the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (sars-cov) in 2002. more than 1500 mers-cov cases were recorded in 42 months with a case fatality rate (cfr) of 40%. meanwhile, 8000 cases of sars-cov were confirmed in six months with a cfr of 10%. the clinical presentation of mers-cov ranges from mild and non-specific presentation to progressive and severe pneumonia. no predictive signs or symptoms exist to differentiate mers-cov from community-acquired pneumonia in hospitalized patients. an apparent heterogeneity was observed in transmission. most mers-cov cases were secondary to large outbreaks in healthcare settings. these cases were secondary to community-acquired cases, which may also cause family outbreaks. travel-associated mers infection remains low. however, the virus exhibited a clear tendency to cause large outbreaks outside the arabian peninsula as exemplified by the outbreak in the republic of korea. in this review, we summarize the current knowledge about mers-cov and highlight travel-related issues. the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (sars-cov) in 2002 [1, 2] . as of march 17, 2016 , 1715 mers-cov cases were confirmed with a case fatality rate (cfr) of approximately 40%, whereas 8000 sars-cov cases were recorded in six months with a 10% cfr [1] [2] [3] . an upsurge in the number of cases was observed in march-may 2014 because of the outbreak in large healthcare facilities in jeddah, kingdom of saudi arabia (ksa) [4, 5] . more recently, a larger outbreak occurred in riyadh, ksa and in multiple hospitals in south korea [6] [7] [8] [9] . wolfe et al. [10] described the five stages of pathogen evolution that lead to diseases confined to humans. in stage 1, the pathogen is confined to an animal host. humans become infected by animals only in stage 2. stage 3 is limited human-to-human transmission. in stage 4, long outbreaks with numerous cycles of human-to-human transmission occur. in stage 5, the pathogen exclusively infects humans [10] . mers-cov has not yet reached stage 5. the interest and concern of the public and the health community in emerging infectious diseases stem from the ability of the emerging pathogens to cause pandemics with high fatality rates and the associated economic effects on affected countries. for example, sars-cov caused $30-50 billions of economic losses in mainland and hong kong of china, singapore, and canada, and the pandemic influenza h1n1 in 2009 resulted in $45-55 billion losses worldwide [11] . in this review, we focus on the important aspects of the recently emerged mers-cov, its effects on humans, and the transmission patterns of the disease based on available scientific data. year-old male from bisha [12, 13] . the patient was hospitalized with community-acquired pneumonia; the disease rapidly progressed, resulting in acute renal failure, respiratory failure, and death [13] . a summary of the major events in the development of the mers-cov epidemic is shown in fig.1 . since the first case of mers-cov, a detailed investigation was conducted to determine the environmental and animal source of the virus. extensive contact investigation was carried out on the first case that originated from bisha but was eventually hospitalized in a private hospital in jeddah [12, 14] . the contact investigation started in the patient's hometown of bisha in southern ksa. the investigation included his immediate household contacts (3 wives, 10 sons, 11 daughters, 12 grandchildren, and 1 house maid), as well as the community and healthcare facility in bisha, which included 2 shepherds who took care of his five camels and 14 healthcare workers (hcws) (11 nurses and three physicians) [14] . in total, 53 contacts in bisha were screened and all of them tested negative for mers-cov by polymerase chain reaction (pcr) [14] . an extensive investigation of the 48 out of 56 hcw who had significant contact with the same patient during his 10-day stay in the private hospital in jeddah tested negative [15] . during the october 2012 investigation on the source of the virus, a team representing three agencies, namely, saudi ministry of health, center for infection and immunity of columbia university, and ecohealth alliance, interviewed the family of the index case-patient from bisha. the team also collected samples from bats within 12 km from his home, as well as an abandoned date palm orchard, the area within 1 km from his place of employment, a hardware store that fronted his garden, and a date palm orchard. although none of his family members nor employees recalled seeing bats, the investigating team observed the roosting bats and guano in abandoned wells and ruins within 12 km of his home and insectivorous bats at dusk in the garden behind his store. a sample from a taphozous perforatus bat (egyptian tomb bat) captured in bisha showed 100% identity to the human β-cov 2c emc/2012 cloned from the index case-patient [16] . the largest data set on the contact investigation revealed that the percentage of positive cases was 2.5%, 1.12%, 3.03%, and 2.09% among hospital patients, hcw contacts, family and contacts, and overall [17] . with the expansion of testing to identify the full disease spectrum and the inclusion by the world health organization (who) of the positive cases by serology in july 2014 [18] , the cfr gradually decreased from 60% to 40% as more asymptomatic and mildly symptomatic cases were included [19] . a nationwide serosurvey for mers-cov conducted in the ksa between december 2012 and december 2013 suggested that around 45 000 infected individuals were not aware of their infection, which confirmed an extremely high incidence of asymptomatic to mildly symptomatic disease [20] . a good example of a family cluster of mers-cov infections was published in the new england journal of medicine [21] . once the second case in that cluster was identified, an epidemiologic investigation identified the index case who was the father of the second case who was admitted earlier with congestive heart failure and community-acquired pneumonia. subsequently, the third and fourth cases where recognized [21] . in that cluster, 28 persons lived in the same extended household, including nine children ( < 14 years of age) [21] . aside from the four patients, no other family members had major respiratory symptoms and none of the 124 hcws who managed the index case before the mers-cov diagnosis exhibited symptoms [21] . a second family cluster was also described by omrani et al. [22] . three patients lived in one large house in urban riyadh. none of the other family contacts had positive pcr tests. community outbreak was also described [23] . the first patient infected his cousin, and each of the patients infected their parents. genome analysis showed multiple introduction of the virus and determined three distinct genotypes, which confirm very low patient-to-patient transmission [23] [24] [25] [26] . a number of healthcare-associated outbreaks were reported previously [14, 19, 27, 28] . a cluster of acute respiratory illnesses was reported in the intensive care unit (icu) in zarqa, jordan in april 2012 [29] . thirteen hcw cases were detected (intensivists and icu nurses) with two mortalities. an initial investigation revealed no etiology. after the announcement of the first case of mers-cov in september 2012 in ksa, samples from these patients were retested for mers-cov and confirmed to be positive in two patients by pcr and eight contacts by serology [29, 30] . thus, this case demonstrated mers-cov as a healthcare outbreak. the second large outbreak occurred in al-hasa, ksa in april 2013 [26] . a total of 23 confirmed cases and 11 probable cases were recorded. a detailed transmission map was drawn based on the best available epidemiological data linking these patients epidemiologically [26] . subsequent genotyping showed multiple introduction of the virus leading to the outbreak rather than a single introduction [25] . four of the cases did not match the transmission, which indicates that the disease was not likely transmitted between the cases but had a different genome, thus indicating multiple community introductions [25] . the outbreak was controlled with simple infection control measures in three weeks. mers-cov was shown to have minor clades, and the most recent common ancestor of mers-cov was introduced into humans at the end of 2010 [31] . mers-cov is closely related to pipistrellus bat cov hku5 (pi-batcov hku5) in bats from hong kong and these bats diverged from a common ancestor several centuries ago [32] . in 2014, one of the largest healthcare-associated outbreaks of mers-cov infection took place between february 17 and april 26 in jeddah, ksa [4] . a total of 128 mers-cov patients were treated in 14 hospitals [4, 5] . the number of cases in each hospital varied from 2 to 45 cases [4, 5] . in all the cases, 33% were primary cases and 60% (including 39 hcw) were healthcare-associated infections [33] . the rapid increase in the cases was attributed to more sensitive case detection, active case determination, and contact tracing with changes in testing algorithms [4] . a breakdown in infection control measures was observed with no change in the virus [4] . a near full genome sequence of the three viruses from the early phase of the jeddah outbreak showed a highly similar virus with no genome insertions or deletions [4] . the genetic marker influencing transmissibility was 100% identical to known mers-cov genomes [4] . in 2015, the largest outbreak outside the middle east took place in south korea [6] . the index case was a 68year-old male who visited bahrain, ksa, united arab emirates, and qatar [6] . he developed symptoms on may 11, 2015 and visited multiple hospitals in south korea [6] . he caused an outbreak involving five health care facilities and 63 cases [7] , with 34 cases in hospital b, 18 cases in hospital d, 5 cases in hospital e, and three cases in hospital f [7] . as of june 19, 2015 the outbreak in the republic of korea involved 72 health care facilities, and six facilities exhibited nosocomial transmission [8] . the total number of cases as of july 21, 2015 was 186 cases with 36 deaths [34, 35] . based on epidemiological data monitoring over the last three years, the potential seasonality of mers-cov from march to may and from september to november was observed. in april and may 2014, the number of cases increased [4] . one of the reasons for this increase in the number of cases is the parallel surge in mers-cov tests in jeddah [4] . this increase is also facilitated by an intensified intra-hospital and inter-hospital transmission of mers-cov with no change in the virus genetic composition or ability to cause disease [4, 27] . thus, seasonality is difficult to establish because sporadic cases were documented with amplifications mainly occurring during nosocomial outbreaks. available data to date show that mers-cov behaves differently in various conditions and in different population of patients. isolated sporadic cases, small family clusters, as well as large healthcare-acquired infections and clusters, were recorded. most cases present with respiratory symptoms and about one-third had gastrointestinal symptoms (table 1) [26, [36] [37] [38] [39] [40] . early symptoms are mild and non-specific, which last several days prior to progressing to severe pneumonia. no predictive signs or symptoms exist to differentiate mers-cov from community-acquired pneumonia in hospitalized patients [38] . an apparent heterogeneity in transmission was observed. the severity of the disease is usually seen in primary or index cases, immune-compromised individuals, and people with underlying comorbidities. mild or asymptomatic disease usually occurs in secondary cases and was initially thought to infect the young and previously healthy individuals. however, mortalities and severe cases were observed among primary cases and young individuals [10] . however, person-to-person transmission as a definite route of transmission is still unclear. the median incubation period was 5.2 days (95% ci, 1.9 to 14.7), and the serial interval was 7.6 days (95% ci, 2.5 to 23.1) [26] . cfr is directly related to the number of comorbidities, the increasing detection of asymptomatic to mildly symptomatic cases over the last three years [39] . however, mortalities were reported among healthy individuals. the median time to hospitalization was 4 days, icu admission was 5 days, mechanical ventilation was 7 days, and death was 11.5 days [26, 41] . mers-cov pcr was standardized, and it works extremely well with lower respiratory samples in experienced laboratories. confirmatory testing in national or regional reference laboratories with experience and load of samples will avoid reporting false positive cases. on november 5, 2013, a spanish case from hajj was initially tested positive for mers-cov but was eventually sent to an outside reference laboratory for confirmatory testing; all tests were negative [42] . if mers-cov infection is suspected and initial testing is negative, repeat testing is recommended and lower respiratory tract samples would yield higher positivity [43] . in july 2014, a key change in mers-cov case definition is the inclusion of a confirmed case based on serology [44] . serologic mers-cov confirmation requires sero-conversion in two samples taken at least 14 days apart by a screening (elisa, ifa) and a neutralization assay [44] . since the emergence of mers-cov and till october 2015, 1715 cases were reported in different countries [45] . the cases included 1403 cases in the middle east, 15 cases in europe, 191 in asia, and 7cases in other countries [45] . the who international health regulations (ihr) emergency committee convened a mers-cov emergency committee meeting on multiple occasions; after extensive deliberations and reviews of available data, the diseases weakness did not fulfill the ihr requirements to be defined as a public health emergency of international concern (pheic) and mainly sustained human-to-human transmission [46, 47] (fig.2) . using a mathematical model, the risk of mers-cov was estimated to be one to seven cases per hajj and three to ten umrah pilgrims per year [48] . in another model, 6.2 pilgrims were estimated to develop mers-cov symptoms during the hajj, and 4.0 foreign pilgrims will be infected but return home before developing symptoms [49] . travelrelated mers-cov occurred infrequently among pilgrims performing the umrah [50] . however, millions of pilgrims who performed the annual hajj did not exhibit mers-cov symptoms [51] . a cross sectional study of 839 african hajj pilgrims returning to ghana, west africa in 2013 showed that none of the pilgrims was positive for mers-cov [52] . a cohort of french pilgrims exhibited no mers-cov infection [53] . no mers-cov was detected by pcr among 5235 adult pilgrims from 22 countries [54] . although the risk of travel-associated mers-cov remains low, the potential for healthcareassociated infections in relation with an imported mers-cov is a real concern. this event took place in the republic of korea [6] [7] [8] [9] . thus, all hcws should be vigilant to the importation of mers from returning travelers and healthcare organizations should implement a strategy to screen, isolate, and diagnose these patients. serologic testing allowed the detection of eight of the 124 contacts in the jordan cluster [30] . the positive results were confirmed in six of nine outbreak members, one in 26 household contacts, and one in 89 hcws [30] . interest-ingly, one hcw who tested positive did not recall having respiratory symptoms at the time of the outbreak [30] . a few published serology studies did not present any background on mers-cov. in one study, eight out of 356 abattoir workers and blood donors had weak positive tests by ifa, and none of these individuals tested positive by nt in jeddah and makkah in 2012 [55] . in a second study, none of 268 children with respiratory tract disease and blood donors showed neutralizing antibodies in dammam, ksa in 2010-2012 [55] . an initial study of 268 samples that were tested for mers-cov antibodies in the eastern saudi arabia revealed no positive samples [55] . a serologic evaluation of 280 household contacts of 26 index patients showed 12 probable cases of secondary transmission (4%; 95% confidence interval, two to seven) [56] . a large-scale study of more than 10 000 samples demonstrated that the overall seroprevalence in ksa was 0.15% and detected an increase of 17-and 26-fold of antibody detection rate among camel shepherds and abattoir workers compared to that in the general population [20] . the full genome sequences from mers patients with known dates and locations can help answer these questions: how fast does the virus change? when did the virus begin circulating in its current form? is the virus adapting to humans? can the geographical patterns help locate an animal source? what are the transmission patterns? a previous study showed that sequence success is as function of viral load, which is inversely proportional to the threshold cycle value (ct) in real-time pcr assays [25] . ct values below 33 are associated with good sequencing success rates. thus, ct values are sufficient predictors of the success that is independent on sample type or source [24, 25] . mers-cov was found to be stable in the environment. the virus can survive on plastic and steel for up to 48 h at lower temperatures and humidity; once temperature and humidity increase, the virus becomes less viable [57] . the virus is viable at 20°c and 40% humidity for 48 hours, at 30°c and 30% humidity for 24 hours and at 30°c and 80% humidity for 8 hours only [57] . in another study, increasing the temperature from 25°c to 65°c adversely affects viral infectivity [58] . the who advocates contact and droplet precautions with airborne isolation in hospital settings when dealing with an aerosol-generating procedure [56] ; the united states centers for disease control and prevention (cdc) and the european centre for disease prevention and control (ecdc) call for airborne infection isolation precautions [59] [60] [61] . multiple hospital outbreaks of mers-cov infection in ksa were controlled effectively using infection control precautions recommended by the who and the saudi ministry of health [4, 5, 26, 36] . there are no proven therapeutic agents for the treatment of mers-cov patients. existing antiviral agents can be repurposed against mers-cov [62, 63] . interferon and ribavirin were suggested to be possible therapeutic options based on the sars data [63] . these two agents were used to treat five patients with mers-cov infection [64] . the median time to therapy was 19 days, and no improvement was observed [64] . ribavirin and interferon were used in 20 patients with mers-cov at a median of three days [65] . the 14-day survival was 70% (14 of 20 patients) in the treatment group vs. 29% (7 of 24 of patients) in a historical group (p = 0.004) with no survival improvement at 28 days (30% vs. 17%; p = 0.054) [63] . in an observational study, interferon-α2a with ribavirin and interferon-β1a with ribavirin showed similar results in treating mers-cov [40] . the potential repurposed drugs for mers therapy include ribavirin with or without interferon, hiv protease inhibitors (lopinavir and nelfinavir), cyclophilin inhibitors (cyclosporin and alisporivir), chloroquine, mycophenolic acid, and nitazoxanide [66] . the use of interferon-α2b and ribavirin decreased viral replication in the rhesus macaques model within 8 h of mers-cov infection [67] . in vitro, ribavirin inhibits mers-cov; however, the required doses are extremely high to obtain in vivo [68, 69] . in vitro, nelfinavir and lopinavir achieved inhibitory concentrations against mers-cov [70] . in a primate model, the mortality rate at 36 h post-inoculation was 67% in untreated versus 0%-33% in the lopinavir-or ritonavirtreated and interferon-β1b-treated animals [71] ; the combination was used in another case [72] . to understand the mers-cov disease further, resolving the issues related to the specific host and the specific transmission mode and determining the factors that increase transmission within healthcare environments are crucial. the viral kinetics of mers-cov within different body compartments is another aspect that needs further examination. the optimal therapeutic options and strategies to predict the occurrence and severity of the disease require further analysis. jaffar a. al-tawfiq, ali s. omrani, and ziad a. memish declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. travel implications of emerging coronaviruses: sars and mers-cov coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers middle east respiratory syndrome coronavirus (mers-cov) -kuwait an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh mers-cov outbreak in jeddah-a link to health care facilities middle east respiratory syndrome coronavirus (mers-cov) -republic of korea south korea coronavirus mers case list-including imported and exported cases middle east respiratory syndrome 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infections: new drug development and therapeutic options treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel coronavirus replication by a combination of interferon-α2b and ribavirin broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-α for middle east respiratory syndrome: a case report key: cord-345046-str19r9a authors: al ghamdi, mohammed; alghamdi, khalid m.; ghandoora, yasmeen; alzahrani, ameera; salah, fatmah; alsulami, abdulmoatani; bawayan, mayada f.; vaidya, dhananjay; perl, trish m.; sood, geeta title: treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia date: 2016-04-21 journal: bmc infect dis doi: 10.1186/s12879-016-1492-4 sha: doc_id: 345046 cord_uid: str19r9a background: middle eastern respiratory syndrome coronavirus (mers-cov) is a poorly understood disease with no known treatments. we describe the clinical features and treatment outcomes of patients with laboratory confirmed mers-cov at a regional referral center in the kingdom of saudi arabia. methods: in 2014, a retrospective chart review was performed on patients with a laboratory confirmed diagnosis of mers-cov to determine clinical and treatment characteristics associated with death. confounding was evaluated and a multivariate logistic regression was performed to assess the independent effect of treatments administered. results: fifty-one patients had an overall mortality of 37 %. most patients were male (78 %) with a mean age of 54 years. almost a quarter of the patients were healthcare workers (23.5 %) and 41 % had a known exposure to another person with mers-cov. survival was associated with male gender, working as a healthcare worker, history of hypertension, vomiting on admission, elevated respiratory rate, abnormal lung exam, elevated alanine transaminase (alt), clearance of mers-cov on repeat pcr polymerase chain reaction (pcr) testing, and mycophenolate mofetil treatment. survival was reduced in the presence of coronary artery disease, hypotension, hypoxemia, cxr (chest x-ray) abnormalities, leukocytosis, creatinine >1 · 5 mg/dl, thrombocytopenia, anemia, and renal failure. in a multivariate analysis of treatments administered, severity of illness was the greatest predictor of reduced survival. conclusions: care for patients with mers-cov remains a challenge. in this retrospective cohort, interferon beta and mycophenolate mofetil treatment were predictors of increased survival in the univariate analysis. severity of illness was the greatest predictor of reduced survival in the multivariate analysis. larger randomized trials are needed to better evaluate the efficacy of these treatment regimens for mers-cov. coronaviruses cause a spectrum of illness from asymptomatic disease to respiratory failure. early reports of coronavirus infections suggested that most infections were mild until the 2003 sars epidemic that was associated with significant morbidity and mortality [1] . in september 2012, a novel coronavirus was identified in a 60-year old man in saudi arabia [2] . a second case was identified in a qatari patient hospitalized in the united kingdom [3] . the two coronaviruses were genetically identical and similar to isolates obtained from bats [4] . in july 2013, the coronavirus study group named this new virus middle east respiratory syndrome coronavirus (mers-cov) [5] . as of december 21, 2015, there have been 1625 cases worldwide with 586 deaths [6] . the epidemiology and clinical manifestations of this disease have described a spectrum of illness from asymptomatic infection to severe respiratory failure and death. the overall mortality rate remains at 37 % [7] [8] [9] [10] [11] [12] [13] [14] [15] . importantly, there are no known effective treatments. in 2014 there was an increase in mers-cov cases reported from the jeddah region of saudi arabia. to describe the changing epidemiology and outcomes, we report the clinical features and treatment outcomes of patients admitted to a regional referral hospital in jeddah, saudi arabia. king fahd general hospital is an 800-bed hospital in jeddah, kingdom of saudi arabia and is a regional coronavirus referral center. there are 36 icu beds and one infectious disease physician that serves the hospital. between january through december 2014, all patients admitted or transferred to king fahd hospital with a positive mers coronavirus pcr from clinical nasal swabs or nasopharyngeal aspirates were included. all pcr testing was performed at the ministry of health regional lab in jeddah. the magna pure compact/ magna pure 96 (roche) automated system was used to extract rna from samples. primers and probes for upe and orf 1a targets of mers-cov were used from tib molbiol (germany) along with master mix from roche for the light cycler 480 ii (roche) were used to amplify upe and orf 1a gene targets. samples that tested positive for both upe and orf 1a gene targets with a cycle threshold time of less than 37 were considered confirmed cases. positive and negative controls were used to monitor the amplification process & to check for any inhibition of amplification. medical charts for all patients were reviewed and data abstracted on standardized data collection forms by an infectious disease trained physician. demographic, clinical and laboratory data were entered into a database. to understand the epidemiology, age was categorized as <30, 30-60 and >60. hypotension was defined as blood pressure <90/60 mm hg, tachypnea as a respiratory rate greater than 16, hypoxia as an oxygen saturation <90 %, thrombocytopenia as platelets <150,000/ cubic millimeter, leukopenia was defined as a white blood cell count <5000 cells/cubic millimeter and leukocytosis as a white blood cell count >10,000 cells/ cubic millimeter. renal insufficiency was defined as a creatinine >1.5 mg/dl. liver function abnormalities were defined as a lactate dehydrogenase (ldh) >300 u/liter, alanine transaminase (alt) > 50 u/liter and aspartate aminotransferase (ast) >40 u/liter. immunosuppression was defined as aids, history of organ transplant, neutropenia, known malignancy, taking immunosuppressive medication and congenital immunodeficiency. pregnancy was considered an immunosuppressed state. a modified acute physiologic and chronic health evaluation (apache 2) score was calculated using age, temperature, mean arterial blood pressure, respiratory rate, potassium, creatinine, acute renal failure, and comorbid conditions to estimate severity of illness [16] . pao2 was estimated using pulse oximetry oxygen saturation results and hematocrit was calculated by multiplying the hemoglobin times three. all statistical analyses were performed using stata software (version 13.1, college station, tx). the percent distribution of clinical variables among patients who survived and those who died were compared using the fisher exact test. a multivariate logistic regression was done on treatments administered and severity of illness to determine which treatments were associated with survival. mycophenolate mofetil was not included in this logistic regression analysis because 100 % of patients receiving mycophenolate mofetil survived. the association between severity of illness and treatments administered was assessed by performing a linear regression of treatments administered onto the modified apache 2 score. there were a total of 51 cases, thirty patients (58.8 %) of whom were saudi nationals, and 21 (41.2 %) were foreign nationals. the median age was 54 years old (iqr 36.5-58). most were male (n = 40, 78.4 %). twenty-one patients (41.2 %) had exposure to a known patient with mers coronavirus and 12 (23.5 %) were healthcare workers. none of the patients had animal exposure. two patients (3.9 %) were on pilgrimage to mecca. overall, 71 % of patient had at least one co-morbid condition. seventeen patients had diabetes (33.3 %), 25 had hypertension (49 %), 14 (27.5 %) had end stage renal disease, eight (15.7 %) had coronary artery disease and six (11.8 %) patients were immunosuppressed, two of whom were pregnant. patients received a variety of novel treatments including immunosuppressants and antivirals. forty-two (82.4 %) patients received broad-spectrum antibiotics and five (9.8 %) received hydrocortisone. thirty one patients received antiviral treatment. twenty-three patients (45.1 %) were treated with interferon beta, eight (15.7 %) were treated with interferon alpha. a variety of anti-viral combinations were used. eight patients (15.7 %) received mycophenolate mofetil, seven of these patients received it in combination with interferon beta. nineteen (37.3 %) patients required intensive care unit (icu) care, and 10 patients received extracorporeal membrane oxygenation (ecmo). all patients treated in the icu and all patients receiving ecmo died. in this recent cohort, when comparing survivors to nonsurvivors, survival was associated with male gender, vomiting on admission, elevated respiratory rate, abnormal lung exam on physical exam, working as a healthcare worker, history of hypertension, elevated alt, clearance of mers cov on repeat pcr testing, and receiving mycophenolate mofetil or beta interferon (table 1 ). in contrast, markers of severe disease like hypotension, hypoxemia, chest radiographic abnormalities, leukocytosis, elevated creatinine, thrombocytopenia, anemia, renal failure were associated with death. treatments given were based as indicated based on the clinical assessment of the infectious disease consult team. thirty-one patients received antivirals, ribavirin or alpha or beta interferon, and 13 patients received immunosuppressive medication. most patients received a combination of alpha interferon and ribavirin (5, 9.8 %), beta interferon and ribavirin (10, 19.5 %) or beta interferon alone (11, 21.6 %). two patients received alpha interferon alone (3.9 %). eight patients received mycophenolate mofetil (15.7 %) and seven of them received this in combination with beta-interferon. five patients received hydrocortisone; two in combination with beta interferon and ribavirin and 3 in combination with alpha interferon and ribavirin. all eight patients given mycophenolate mofetil survived therefore mycophenolate mofetil could not be evaluated in this model. while the results of the univariable analysis demonstrated improved survival in patients treated with betainterferon and mycophenolate mofetil, the multivariable analysis which included a marker of severity of illness, demonstrated a strong association between severity of illness and reduced survival, and no association between treatment with beta interferon and survival. mycophenolate mofetil was not evaluable in this model ( table 2 ). in analyzing the relationship between severity of illness and treatments administered, beta interferon and mycophenolate mofetil were given to less severely ill patients (table 3) discussion mers-cov is an emerging disease for which the initial epidemiology has been described, but in-depth clinical studies and the role of therapy in incompletely understood. while the clinical features for mers-cov have been described in several large case series [6] [7] [8] [9] [10] [11] [12] [13] [14] , there is a paucity of literature on therapy. our results from a relatively large number of patients demonstrate similar clinical features and mortality to previous studies [6] [7] [8] [9] [10] [11] [12] [13] [14] . in our cohort, treatment with beta interferon and mycophenolate mofetil may be predictive of survival, but the greatest predictor of survival is the severity of illness on presentation. improved diagnostics have demonstrated an expanded spectrum of disease that includes less severe cases than previously reported. we now understand that mers-cov causes an acute respiratory disease syndrome and [7] [8] [9] [10] [11] [12] [13] [14] [15] . this may be partially related to the epidemiology of increased disease transmission in healthcare settings rather than a true host risk factor. laboratory findings have been nonspecific and consistent with other viral infections. thrombocytopenia (75 %) and lymphopenia (58 %) have been commonly described in these patients [7, [9] [10] [11] [12] [13] 15] . forty three percent had acute kidney injury [7, [11] [12] [13] 17] and 76-100 % had cxr abnormalities with bibasilar infiltrates as the most common finding [8-13, 15, 18] . the outcomes in these more severely ill patients remain poor. between 50-90 % required icu care [10, 11, 13, 15] and 67-100 % in the icu setting required invasive ventilation for a median of 7-16 days [8, 10, 12] . in addition to mechanical ventilation, several patients have received extracorpeal membrane oxygenation (ecmo) to support ventilation. from non-randomized data from the world health organization, five out of six patients receiving ecmo died [9] . fifty-eight to 75 % required renal replacement therapy [11, 12, 17] and 30-60 % of hospitalized patients died [7] [8] [9] [10] [11] [12] [13] [14] [15] . the severity of illness can be partially explained by the widespread lung disease caused by mers-cov and it appears that mortality in those patients requiring intensive care is extremely high. although no autopsy data is available, in explanted lung, infection with mers-cov causes widespread infection and alveolar disease [19, 20] . the clinical features in our cohort similarly also show a high proportion of patients with fever (96 %) and cough (80.4 %) shortness of breath (90 %), and almost one third of patients (29.4 %) with gastrointestinal symptoms. our cohort consisted of ill patients with hypotension (15.7 %), tachypnea (76.9 %) and hypoxia (33 %). thirty seven percent required icu care and 10 patients received ecmo. similar to previous results, all of the patients who received ecmo died [9] . there is no known effective treatment for mers cov. many compounds have been screened in vitro for possible activity against this coronavirus [21] [22] [23] [24] , however, the in vivo efficacy has not been subjected to clinical investigation. in vitro data suggests that mers-cov inhibits host interferon production through various molecular pathways [25] [26] [27] [28] [29] [30] mycophenic acid, the active agent of prodrug mycophenolate mofetil, and cyclosporine strongly inhibit mers coronavirus in human and monkey cell lines even more so than they inhibit sars coronavirus [24, [31] [32] [33] . interferon alpha and interferon beta reduce mers coronavirus replication in explanted lung tissue [19] . in vivo, comparing host response in two patients with mers coronavirus and differing outcomes, the patient who was able to clear mers cov infection was able to mount an interferon response and the patient who died had low levels of interferon alpha suggesting a therapeutic role for interferon [34] . the combination of interferon alpha and ribavirin has been used successfully in rhesus monkeys infected with mers coronavirus [35] , and in a few small case series [36] [37] [38] . beta-interferon seems to be an even more potent inhibitor of mers coronavirus in vitro [19] [24, [31] [32] [33] . one small study with exceptionally high mortality rates using interferon beta for treatment found no difference in mortality between interferon beta use and interferon alpha use [39] . our data, albeit from a retrospective cohort support the findings that interferon beta is associated with a decrease in mortality. there are limited data on the efficacy of treatment regiments for this virulent disease. we present data from a retrospective cohort of ill patients with mers-cov and the results of the evaluation of the clinical efficacy of beta interferon beta, alpha interferon, ribavirin and mycophenolate mofetil in addition to routine supportive care. forty five percent of patents (23 patients) received interferon beta and in this cohort, sixteen percent of patients received interferon alpha (8 patients) and 37 % of patients (9 patients) received ribavirin, either in conjunction with interferon alpha or interferon beta, and 8 patient received mycophenolate mofetil. patients receiving beta interferon and mofetil had improved survival, however this was confounded by the severity of illness on presentation for beta interferon. all of the patients who received mycophenolate mofetil survived however because of the small number, we could not analyze the independent efficacy of mycophenolate mofetil. while this is a relatively large series of mers-cov cases, the primary limitation of our study is that it is a retrospective review of cases and not a randomized trial and thus subject to confounding as seen in our cohort. we used a modified apache 2 score without all of the clinical variables, which may have underestimated the association of severity of illness with reduced survival. importantly, the mortality in patients receiving additional therapies that modulate the immune response was low. all of the eight patients who received mycophenolate mofetil in our study survived. hence, it may be reasonable to further study this agent in controlled trials. this observational study investigates novel treatment options like beta interferon and mycophenolate mofetil for mers-cov in humans which have in vitro activity. our cohort demonstrated severity of illness is an important effect modifier and needs to be considered in evaluating novel agents. to better assess the efficacy of these therapies, international prospective randomized trials with adequate numbers of patients are needed to further evaluate the impact of these treatments in addition to routine supportive care when compared to other treatment options. this study was reviewed and approved by johns hopkins university institutional review board and the directorate of health affairs. data supporting the findings are in the manuscript, additional data available upon request. abbreviations aids: acquired immune deficiency syndrome; alt: alanine transaminase; apache 2: acute physiologic and chronic health evaluation; ast: aspartate aminotransferase; cxr: chest x ray; ecmo: extracorporeal membrane oxygenation; icu: intensive care unit; ldh: lactate dehydrogenase; mers co-v: middle eastern respiratory syndrome coronavirus; pcr: polymerase chain reaction. the authors declare that they have no competing interests. authors' contributions ma conceived of the study, participated in its design and helped draft the manuscript. ka participated in data collection and analysis, and reviewed the manuscript. yg participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. fs participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. mb participated in data collection and analysis, and reviewed the manuscript. tmp participated in the design and analysis as well as the writing of the manuscript. dv participated in the statistical analysis of the study. gs helped analyze the data and write the manuscript. all authors read and approved the final manuscript. the severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia patient with new strain of coronavirus is treated in intensive care at london hospital latest outbreak news from promed-mail: novel coronavirus -middle east middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group who summary family cluster of middle east respiratory syndrome coronavirus infections ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection jordan mers-cov investigation team. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients apache ii: a severity of disease classification system in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses screening of an fda-approved compound library identifies four smallmolecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus molecular pathology of emerging coronavirus infections middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon (ifn)-alpha-2b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study there are no acknowledgements. there was no external funds provided for this project. key: cord-266260-t02jngq0 authors: ramshaw, rebecca e.; letourneau, ian d.; hong, amy y.; hon, julia; morgan, julia d.; osborne, joshua c. p.; shirude, shreya; van kerkhove, maria d.; hay, simon i.; pigott, david m. title: a database of geopositioned middle east respiratory syndrome coronavirus occurrences date: 2019-12-13 journal: sci data doi: 10.1038/s41597-019-0330-0 sha: doc_id: 266260 cord_uid: t02jngq0 as a world health organization research and development blueprint priority pathogen, there is a need to better understand the geographic distribution of middle east respiratory syndrome coronavirus (mers-cov) and its potential to infect mammals and humans. this database documents cases of mers-cov globally, with specific attention paid to zoonotic transmission. an initial literature search was conducted in pubmed, web of science, and scopus; after screening articles according to the inclusion/exclusion criteria, a total of 208 sources were selected for extraction and geo-positioning. each mers-cov occurrence was assigned one of the following classifications based upon published contextual information: index, unspecified, secondary, mammal, environmental, or imported. in total, this database is comprised of 861 unique geo-positioned mers-cov occurrences. the purpose of this article is to share a collated mers-cov database and extraction protocol that can be utilized in future mapping efforts for both mers-cov and other infectious diseases. more broadly, it may also provide useful data for the development of targeted mers-cov surveillance, which would prove invaluable in preventing future zoonotic spillover. middle east respiratory syndrome coronavirus (mers-cov) emerged as a global health concern in 2012 when the first human case was documented in saudi arabia 1 . now listed as one of the who research and development blueprint priority pathogens, cases have been reported in 27 countries across four continents 2 . imported cases into non-endemic countries such as france, great britain, the united states, and south korea have caused secondary cases [3] [4] [5] , thus highlighting the potential for mers-cov to spread far beyond the countries where index cases originate. reports in animals suggest that viral circulation could be far more widespread than suggested by human cases alone [6] [7] [8] . to help prevent future incidence of mers-cov, public health officials can focus on mitigating zoonotic transfer; however, in order to do this effectively, additional research is needed to determine where spillover could occur between mammals and humans. previous literature reviews have looked at healthcare-associated outbreaks 9 , importation events resulting in secondary cases 10, 11 , occurrences among dromedary camels 12, 13 , or to summarize current knowledge and knowledge gaps of mers-cov 14, 15 . this database seeks fill gaps in literature and build upon existing notification data by enhancing the geographic resolution of mers-cov data and providing occurrences of both mammal and environmental detections in addition to human cases. this information can help inform epidemiological models and targeted disease surveillance, both of which play important roles in strengthening global health security. knowledge of the geographic extent of disease transmission allows stakeholders to develop appropriate emergency response and preparedness activities (https://www.jeealliance.org/ global-health-security-and-ihr-implementation/joint-external-evaluation-jee/), inform policy for livestock trade and quarantine, determine appropriate demand for future vaccines (http://cepi.net/mission) and decide where to deliver them. additionally, targeted disease surveillance will provide healthcare workers with updated lists of the methods and protocols summarized below have been adapted from previously published literature extraction processes [18] [19] [20] [21] [22] , and provide additional context surrounding our systematic data collection from published reports of mers-cov. data collection. we identified published reports of mers-cov by searching pubmed, web of science, and scopus with the following terms: "middle eastern respiratory syndrome", "middle east respiratory syndrome", "merscov", and "mers". the initial search was for all articles published about mers-cov prior to april 30, 2017 , and was subsequently updated to february 22, 2018. these searches were conducted through the university of washington libraries' institutional database subscriptions. we searched the web of science web of science core collection (the subscribed edition includes science citation index expanded, 1900-present; social sciences citation index, 1975-present; arts & humanities citation index, 1975-present; emerging sources citation index, 2015-present). we searched the standard scopus database and the standard, freely available pubmed database; these products have a single version that is consistent across institutional subscriptions or access points. in total, this search returned 7,301 related abstracts, which were collated into a database before a title-abstract screening was manually conducted (fig. 1. flowchart) . articles were removed if they did not contain an occurrence of mers-cov; for example, vaccine development research or coronavirus proteomic analyses. non-english articles were flagged for further review and brought into the full text screening stage. the accompanying supplementary file highlight the title and abstract screening process and the inclusion and exclusion criteria. full text review was conducted on 1,083 sources. to meet the inclusion criteria, articles must have contained both of the following items: 1) a detection of mers-cov from humans, animals, or environmental sources, and 2) mers-cov occurrences tagged with spatial information. additionally, extractors attempted to prospectively manually remove articles containing duplicate occurrences that were already extracted in the dataset. extractors only prospectively manually removed articles if it was clear the articles contained data we were confident had already been extracted and had high-quality data. we excluded 885 sources based on full text review. in addition, we reviewed citations and retroactively added relevant articles to our database if they were not already included. we retroactively added and subsequently marked ten articles for extraction using this process. in total, we extracted 208 peer-reviewed sources reporting detection of mers-cov that included geographic and relevant epidemiological metadata. geo-positioning of data. google maps or arcgis 23 was used to manually extract location information at the highest resolution available from individual articles. we evaluated spatial information as either points or polygons. the geography was defined as a point if the location of transmission was reported to have occurred within a 5 × 5 km area. point data are represented by a specific latitude and longitude. a point references an area smaller than 5 × 5 km in order to be compatible with the typical 5 × 5 km resolution of satellite imagery used for global analyses. the geography was defined as a polygon if the location of transmission was less clear, but known to have occurred in a general area (e.g. a province), or the location of transmission occurred within an area greater than 5 × 5 km (e.g. a large city). we used contextual information to determine location in instances where the author's spelling of a location differed from google maps or arcgis. maps provided by authors were digitized using arcgis. we used three different types of polygons: known administrative boundaries, buffers, and custom polygons. relevant administrative units were sourced from the global administrative unit layers curated by the food and agricultural organization of the un 24 for known administrative boundaries of governorates, districts, or regions, and paired with the occurrence record. buffers were created to encompass areas in cities and regions without corresponding administrative units. to ensure that buffers encompassed the entirety of the area of interest, google maps was used to determine the required radius. in areas with unspecified boundaries (e.g. table mountain national park and the border region between saudi arabia and uae) arcgis was used to generate custom polygons, which were assigned a unique code within a defined shapefile for ease of re-identification. this database is publicly available online 16, 17 . each of the 861 rows represents a unique occurrence of mers-cov. rows containing an index, unspecified, or imported case represent a single case of mers-cov. rows containing mammal and secondary cases may represent more than one case but are still unique geospatial occurrences. table 1 shows an overview of the content available in the publicly available dataset. in addition, online-only table 1 lists occurrences by geography, origin, 405 shape type, and publication and online-only table 2 provides citations of the data. index 34 99 1 0 93 7 234 unspecified 86 50 1 4 35 27 203 mammal 53 56 7 30 43 19 208 import 11 2 0 2 10 9 34 secondary 82 30 1 1 26 8 148 absent 3 8 0 0 7 3 21 environmental www.nature.com/scientificdata www.nature.com/scientificdata/ 15. pathogen: name the pathogen identified (e.g. mers-cov, bat coronaviruses, and other mers-cov-like pathogens). 16. pathogen_note: miscellaneous notes regarding pathogen. 17. patient_type: index, unspecified, na, secondary, import, or absent. • index: any human infection of mers-cov resulting after direct contact with an animal and no reported contact with a confirmed mers-cov case or healthcare setting. • unspecified: cases that lacked sufficient epidemiological evidence to classify them as any other status (e.g. serosurvey studies). • na: non-applicable field; case was not a patient (e.g. mammal) • secondary: defined as any cases resulting from contact with known human infections. cases reported after the index case can be assumed to be secondary cases unless accompanied by specific details of likely independent exposure to an animal reservoir. • import: cases that were brought into a non-endemic country after transmission occurred elsewhere. • absent: suspected case(s) ultimately confirmed negative for mers-cov. 18 . transmission_route: zoonotic, direct, unspecified, or animal-to-animal. • zoonotic: transmission occurred from an animal to a human. • direct: only relevant for human-to-human transmission. • unspecified: lacked sufficient epidemiological evidence to classify a human case as zoonotic or direct. • animal-to-animal: transmission occurred from an animal to another animal. 19 . clinical: describes whether the mers-cov occurrence demonstrated clinical signs of infection. denoted by yes, no, or unknown. • yes: clinical signs of infection were present/reported. clinical signs among humans may range from mild (e.g. fever, cough) to severe (e.g. pneumonia, kidney failure). clinical signs among camels include nasal discharge. • no: clinical signs of infection were not present/reported. • unknown: subject(s) may or may not have been demonstrating clinical signs of infection. for example, some authors did not explicitly mention symptoms, but individuals reportedly sought medical care. another example being when a diagnostic serosurvey was conducted during an ongoing outbreak. the term "unknown" was used when articles lacked sufficient evidence for extractors to definitively label as "yes" or "no". 20. diagnostic: describes the class of diagnostic method that was used. pcr, serology, or reported. 21 . diagnostic_note: more detailed information related to the specific test used (e.g. rk39, igg, or igm serology). 22. serosurvey: describes the context if serological testing was used. • diagnostic: testing of symptomatic patients. • exploratory: historic exposure determined among healthy asymptomatic individuals. 23. country: iso3 code for country in which the case occurred. 24 . origin: open-ended field to provide more details on the specific in-country location of mers-cov case. 25. problem_geography: this field was utilized if the mers-cov case was reported in a location that could cause uncertainty when determining exact geographic occurrence (e.g. hospital, abattoir). 26. lat: latitude measured in decimal degrees. 27. long: longitude measured in decimal degrees. 28. latlong_source: the source from which latitude and longitude were derived. 29. loc_confidence: states the level of confidence that researchers had when assigning a geographic location to the mers-cov case (good or bad). an answer of 'good' meant the article stated clearly that the case occurred in a specific geographic location and no assumptions were required on part of the researcher. an answer of 'bad' meant the article did not clearly state the specific geographic location of the mers-cov case, but the researcher was able to infer the location of occurrence. the field site_notes was utilized to detail the logic behind researchers' decisions when inference was required. 30. shape_type: the geographic shape type assigned to the mers-cov occurrence (point or polygon). 31. poly_type: if the mers-cov occurrence was assigned a shape_type of polygon, was it admin (gaul), custom, or buffer? 32. buffer_radius: if a mers-cov occurrence was assigned a buffer, what is the radius in km? 33. gaul_year_or_custom_shapefile: file path used to reach the necessary shape file in arcgis. users of this dataset can find custom shapefiles created for this dataset at: https://cloud.ihme.washington.edu/index. php/s/dgoykyqnbjg54f2/download 34. poly_id: a standardized and unique identifier assigned to each gaul shapefile. 35 . poly_field: which type of polygon was used to geo-position the occurrence? (e.g. if admin1 polygon was used, enter adm1_code) 36. site_notes: miscellaneous notes regarding the site of occurrence. 37. month_start: month that the occurrence(s) began. if the article provided a specific month of illness onset, the month was assigned a number from 1-12 (1 = january, 2 = february, etc.). if the article did not provide a specific month of illness onset, then researchers assigned a value of 'na' . month that the occurrence(s) ended, defined as the date a patient tested negative for mers-cov. if the article provided a specific month for recovery, the month was assigned a number from 1-12 (1 = january, 2 = february, etc.). if the article did not provide a specific month of symptom onset, then researchers assigned a value of 'na' . 39. year_start: year that the occurrence(s) began. if the year of illness onset was not provided in the article, the ihme standard was used: (year_start = publication year -3). year that the occurrence(s) ended. if the article did not provide a specific year for recovery, the ihme standard was used: (year_end = publication year -1). 41. year_accuracy: if years were reported, this field was assigned a value of '0' . if assumptions were required, this field was assigned a value of '1' . all data extracted from the original search (october 2012 to april 30, 2017) was reviewed independently by a second individual to check for accuracy. challenging extractions from the updated search (may 1, 2017 to february 22, 2018) were selected for group review during bi-weekly team meetings. upon extraction completion, all data were checked to ensure they fell on land and within the correct country. while the protocol implemented above was designed to reduce the amount of subjective decisions made by extractors, total elimination was not possible. wherever a subjective decision had to be made, the extractor utilized the various notes fields in order to document the logic behind decisions. these decisions were subsequently reviewed by other extractors. the techniques described here can be applied to collect and curate datasets for other infectious diseases, as has been previously demonstrated with dengue 20 and leishmaniasis 18 . additionally, since these data were collected independently through published reports of mers-cov occurrence, they may be used to build upon existing notification data 26, 27 . our ability to capture occurrences in this dataset is contingent on the data contained within published literature. therefore, this dataset does not represent a total count of all cases. instead, this dataset's value lies within its geo-precision. data were extracted with a focus on obtaining the highest resolution possible. these data may be merged with other datasets, such as who 26 or oie 27 surveillance records, and are intended to complement, not replace, these resources. together, published reports and notification data can provide a more comprehensive snapshot of current disease extent and at-risk locations. an important consideration, whether using the literature data alone, or in combination with other databases, is the potential for duplication. various pieces of metadata can be used to evaluate where potential duplicates could lie, such as common date fields (month_start, month_end, year_start, year_end) or consistent geographic details (lat, long, poly_id, shape_type) or shared epidemiological tags (patient_type). researchers may wish to consider further steps, such as fuzzy matching of geographic data (e.g. matching a point with an overlapping buffer) or temporal data (e.g. matching a precise month with an overlapping month interval). we acknowledge this duplicate-removal process will not catch all matching records, but it will likely catch several. we recommend occurrences are layered from top to bottom in the following order: index (green), unspecified (orange), mammal (yellow), import (blue), secondary (purple). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this approach because it will allow researchers to remove several duplicates without erroneously deleting any two occurrences that are truly unique (i.e. not duplicates). essentially, we recommend a sensitive approach above a more specific approach, as the latter simply risks culling too many records that aren't actually duplicates. when merging with other databases, consistency in metadata tagging is essential. for the who disease outbreak news data feed 26, 27 for instance, nomenclature for case definitions is slightly different, with who definitions of "community acquired" and "not reported" comparable to "index" and "unspecified" respectively. in addition, it is important to recognize what information is beyond the scope of these additional databases. again, when comparing to the who dataset, it is important to recognize that serologically positive cases do not meet the case definition used in the who database. these adjustments need to be identified on a dataset-to-dataset basis. among cases tagged as index or unspecified. occurrences tagged as index are coloured green, those tagged as unspecified are coloured orange. points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this database can be combined with other covariates (e.g. satellite imagery) to produce environmental suitability models of mers-cov infection risk and potential spillover on both global and regional scales as achieved with other exemplar datasets [28] [29] [30] [31] . this information can be useful in resource allocation aimed at improving disease surveillance and contribute towards a better understanding of the factors facilitating continued emergence of index cases. the addition of sampling techniques and prevalence data may improve this dataset. researchers were ultimately unable to add these data due to inconsistencies in the way literature reported sampling techniques and prevalence date by geography. an attempt to extract these data using the current approach would have led to sporadic inclusion of this information and would not have been comprehensive for the entire dataset. moving forward, we recommend authors report sampling technique and prevalence data at the highest resolution geography possible, as seen in miguel et al. 32 . we encourage continued presentation of paired epidemiological and geographic metadata that would allow for more detailed analysis in the future. this database may also be utilized in clinical settings to provide an evidence-base for diagnoses when used in conjunction with patient travel histories. additionally, it can be used to identify geographies for surveillance, particularly areas where mers-cov has been documented in animals but not humans (e.g. ethiopia and nigeria). identifying locations for surveillance will, in turn, inform global health security. while models will increase the resolution at which these questions can be addressed, datasets such as this provide an initial baseline. a major limitation of this database is the potential for sampling bias, which stems from higher frequency of disease reporting within countries where there exists strong healthcare infrastructure and reporting systems. this database does not attempt to account for such biases, which must be addressed in subsequent modelling activities where such biases are of consequence. similarly, another limitation is potential duplicate documentation of singular occurrences. this can happen when the same occurrence is assigned different geographies (e.g. point, polygon) in multiple publications. even though extractors made efforts to prospectively manually identify duplicate occurrences, this was challenging because the process relied upon papers providing sufficient details for extractors to determine a duplicate occurrence (e.g. geography, patient demographics, dates of occurrence, diagnostic methods, etc.). however, the majority of papers did not report such details for each occurrence. in those instances, it was impossible for extractors to discern whether occurrences may have been duplicates from a previous artic le. even case studies inconsistently reported patient details and demographic information. these are some examples of challenges faced by extractors when we attempted to identify duplicates. without sufficient contextual clues, extractors lacked evidence to determine duplicity and thus likely extracted some unique occurrences more than once. despite efforts to remove duplicate occurrences from the database, it is possible that some remain. geographic uncertainty is similarly problematic for analyses such as this. in some cases, polygons, as opposed to points, are utilised as a geographic frame of reference, reflecting the uncertainty in geotagging in the articles themselves. for some occurrences, there is a strong assumption that the geography listed corresponds to the site of infection. while the use of 5 km × 5 km as the minimum geographical unit allows for some leeway in this precision, it is possible that even with the point data (often corresponding to household clusters) these may not map directly with true infection sites. this must be considered in any subsequent geospatial analysis. finally, this database represents a time-bounded survey of the literature. while all efforts were made to be comprehensive within this period, articles, and therefore data, will continue to be published. efforts to streamline ongoing collection processes are still to be fully realized 33 . regardless, we hope that this dataset provides a solid baseline for further iteration. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission enhanced mers coronavirus surveillance of travelers from 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imported case of middle east respiratory syndrome coronavirus (mers-cov don't forget the migrants": exploring preparedness and response strategies to combat the potential spread of mers-cov virus through migrant workers in sri lanka high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis arabia: an index case investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan stillbirth during infection with middle east respiratory syndrome coronavirus antibodies against mers coronavirus in dromedary camels mers-cov antibodies in humans, africa no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response dromedary camels in northern mali have high seropositivity to mers-cov middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels the middle east respiratory syndrome coronavirus (mers-cov) zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae imported case of middle east respiratory syndrome coronavirus (mers-cov) infection from oman to thailand serologic evidence for mers-cov infection in dromedary camels contact tracing the first middle east respiratory syndrome case in the philippines effectiveness of the middle east respiratory syndrome-coronavirus protocol in enhancing the function of an emergency department in qatar high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases isolation of mers coronavirus from a dromedary camel mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels occupational exposure to dromedaries and risk for mers-cov infection risk factors for primary middle east respiratory syndrome 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coronavirus in al-ahssa region, saudi arabia antibody response and disease severity in healthcare worker mers survivors brief report: family cluster of middle east respiratory syndrome coronavirus infections characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a singlecenter experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia descriptive epidemiology and characteristics of confirmed cases of middle east respiratory syndrome coronavirus infection in the makkah region of saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study evidence for camel-to-human transmission of mers coronavirus exposures among mers case-patients first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities hospital outbreak of middle east respiratory syndrome coronavirus human infection 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syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia outbreak of middle east respiratory syndrome at tertiary care hospital patient characteristics infected with middle east respiratory syndrome coronavirus infection in a tertiary hospital predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients report of middle east respiratory syndrome coronavirus (mers-cov) infection in four patients with hematological malignancies treated 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coronaviruses and isolation of a novel clade a strain from dromedary camels in the united arab emirates prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate time course of mers-cov infection and immunity in dromedary camels transmission of middle east respiratory syndrome coronavirus infections in healthcare settings response to emergence of middle east respiratory syndrome coronavirus diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates mers-cov in pregnancy some epidemiological studies on mers coronavirus in dromedaries in the united arab emirates -a short communication emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives melinda gates foundation opp#1181128 and s.i.h. was supported by opp1132415 curated and catalogued the database. s.s. provided managerial support. all authors participated in interpreting and summarizing the results. r.e.r. wrote the first draft of the manuscript. all other authors critically reviewed the manuscript had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis s.i.h. and d.m.p. are members of the editorial board of scientific data. supplementary information is available for this paper at https://doi.org/10.1038/s41597-019-0330-0.correspondence and requests for materials should be addressed to d.m.p.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.the creative commons public domain dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files associated with this article. key: cord-351186-llnlto7p authors: park, yong-shik; lee, changhwan; kim, kyung min; kim, seung woo; lee, keon-joo; ahn, jungmo; ki, moran title: the first case of the 2015 korean middle east respiratory syndrome outbreak date: 2015-11-14 journal: epidemiol health doi: 10.4178/epih/e2015049 sha: doc_id: 351186 cord_uid: llnlto7p this study reviewed problems in the prevention of outbreak and spread of middle east respiratory syndrome (mers) and aimed to provide assistance in establishing policies to prevent and manage future outbreaks of novel infectious diseases of foreign origin via in-depth epidemiological investigation of the patient who initiated the mers outbreak in korea, 2015. personal and phone interviews were conducted with the patient and his guardians, and his activities in saudi arabia were investigated with the help of the saudi arabian ministry of health. clinical courses and test results were confirmed from the medical records. the patient visited 4 medical facilities and contacted 742 people between may 11, 2015, at symptom onset, and may 20, at admission to the national medical center; 28 people were infected and diagnosed with mers thereafter. valuable lessons learned included: (1) epidemiological knowledge on the mers transmission pattern and medical knowledge on its clinical course; (2) improvement of epidemiological investigative methods via closed-circuit television, global positioning system tracking, and review of health insurance review and assessment service records; (3) problems revealed in the existing preventive techniques, including early determination of the various people contacted; (4) experiences with preventive methods used for the first time in korea, including cohort quarantine; (5) reconsideration of the management systems for infectious disease outbreaks across the country, such as this case, at the levels of central government, local government, and the public; (6) reconsideration of hospital infectious disease management systems, culture involving patient visitation, and emergency room environments. after landing in korea in may of 2015, middle east respiratory syndrome (mers) resulted in a total of 186 confirmed cases and 36 deaths from may 11, when the symptoms occurred in the first patient, to july 4, when the last patient developed the symptoms [1] . a novel coronavirus confirmed for the first time in 2012 is suspected to be the cause of mers and camels may be the host. human-to-human transmission is known to occur after transmission from a camel to a human, but the exact route has not been sufficiently investigated [2] . the outbreak in korea started with a patient who arrived in may 2015 from saudi arabia, a country that had an outbreak of a large number of mers cases, and subsequently spread. a majority of infected patients were revealed to be cases of hospital-acquired infection [2, 3] . this report is an epidemiological study of the first patient (patient #1) who initially brought mers into korea in 2015. from may 20, when a diagnosis of mers was confirmed, the division of epidemic intelligence service, korea centers for disease control and prevention (kcdc), in cooperation with a group of civilian volunteers in epidemiology, traced the infection route and performed preventive measures for the spread of additional infections. through personal and phone interviews we contacted employees at business facility in saudi arabia who may have had contact with patient #1 during the incubation period; we investigated the places he visited, presence or absence of mers symptoms in the individuals he contacted, history of visiting medical facilities in the middle east, and history of consuming camel milk or meat, among other things. the patient's specific activities in saudi arabia were verified with the help of the saudi arabian ministry of health. additionally, the timing of symptom occurrence and the initial symptoms were reevaluated by confirming the history of visiting domestic medical facilities after arrival in korea via personal interviews with the patient and his guardians and examining his medical records. the kcdc performed a diagnostic serum antibody test for mers for individuals whose contact with patient #1 was confirmed by personal interviews or closed-circuit television (cctv) reviews. this study was conducted as an epidemiological investigation of the mers outbreak, and thus, additional processes for review and approval by institutional review board were not required on the basis of the life ethics and safety law enforcement rule item 2 (human subject studies). patient #1 was a 68-year-old man and had underlying diseases including asthma, hypertension, dyslipidemia, and benign prostate hypertrophy. at the time of the study, he was a current smoker. he was in the greenhouse building business with a business facility in the middle east, specifically bahrain, as well as at a domestic business facility in asan, chungnam, korea. he visited the middle east region about once every 2 months and stayed for approximately 3 weeks during each visit. the most recent business trip was between april 24, and may 4, 2015, for 11 days. he had business visits to saudi arabia (may 1 to 2) and the united arab emirates (april 29 to 30) with bahrain as the base; both departure from and arrival at korea was via qatar. during his stay in the middle east, he had no history of direct contact with camels, consuming camel by products such as milk or meat, or visiting the medical facilities there. in saudi arabia, he stayed at his business facility and the hotel in the area of al muzahimiyah outside of the capital, riyadh. during his trips within the middle east, he had no contact with animals and did not eat or drink outside the hotels where he stayed. while staying in riyadh, he travelled with a driver, a guide, and the bahrain business facility manager, none of whom showed the symptoms suspected of mers. patient #1 returned to korea without any abnormal symptoms on may 4 via flight oz6888 and went to his domestic business place in asan on may 11. on may 11, fever broke for the first time, and he visited asan seoul clinic the next day (may 12). at the time of the visit, his body temperature was 37.0°c, and he complained of febrile sense and myalgia. on may 14, he visited the same place with a persistent high temperature (38.9°c) and myalgia, and on may 15, he was transferred to pyeongtaek st. mary's hospital for inpatient treatment, because respiratory symptoms such as cough and sputum had developed, his body temperature was 38.3°c, and the myalgia had worsened. after the transfer, he was in an outpatient exam room, a laboratory room, and a chest radiography room, and was put in a double-occupancy room at approximately 2 pm (room 8104, 8th floor). around 7:15 pm, a chest computed tomography (ct) scan was performed on the first underground floor. on the following day (may 16) at approximately 7:15 am, he visited the radiography room on the first underground floor to undergo a chest radiogram. pneumonia in the right upper lobe was found on the chest ct scan, and infections with haemophilus influenzae and streptococcus pneu monia were confirmed on bacterial culture testing. from the evening of may 15, breathing difficulty and chest pain developed. symptoms did not improve, and the patient was discharged from the hospital at 10 am on sunday, may 17; thereafter, his wife drove him for treatment to the 365 clinic, where he usually went for examination. the 365 clinic referred him to a high-level medical center. he went to the samsung medical center in seoul emergency department, but returned home because a patient room was not available. on the following day (may 18) at approximately 10 am, his wife drove him again to the samsung medical center in seoul emergency department, and he was admitted. from 2 pm, he wore a facial mask. on may 19, he was reported to the kcdc as a case suspected of mers in consideration of the clinical courses that deteriorated despite the antibiotic therapy and his history of travel to the middle east within the previous two weeks, which was revealed during a consultation with physician. on may 20, his sputum polymerase chain reaction (pcr) test was determined positive, following which he was transferred to the national medical center. patient #1 received antiviral, interferon, and antibiotic treatments, and me chanical ventilation therapy after an endo-tracheal intubation was performed due to worsening respiratory symptoms. on june 30, a sputum pcr test was confirmed negative for mers virus, and it was decided that he was cured. on september 25, he was discharged after completing treatment for a sacral sore (figure1). individuals who made contact with patient #1 and preventive efforts for them individuals who were in direct contact with patient #1 without appropriate personal protective equipment or who stayed with him in an enclosed space (patient room, office, etc.) were considered cases of close contact and were put under surveillance. the scope of close contact surveillance gradually expanded with the spread of the mers outbreak, and the final count of individuals under close contact surveillance was 742: 4 employees at patient #1's domestic business facility in asan, 4 medical staff members at the asan seoul clinic, 672 medical staff members and patients (including patient #1's wife) at pyeongtaek st. mary's hospital, 39 medical staff members and patients at the 365 clinic, and 23 medical staff members and patients at the samsung medical center in seoul. depending on the extent of contact, they were classified into either 1) quarantine of a person or a place or 2) active observational surveillance and were closely tracked and observed ( figure 1 ). during surveillance, when symptoms suspected as mers occurred, respiratory samples were collected for testing, the suspected cases were transferred to a quarantined hospital, and epidemiological investigations were conducted. a total of 742 people had contact with patient #1 from may 11, when the first symptoms occurred, to may 20, when he was transferred to the national medical center. of those, 28 became infected, with an infection rate of 3.8%. according to the characteristics of the contacted individuals, 4 out of 235 medical staff members (1.7%); 11 out of 206 patients (5.3%); and 13 out of 301 guardians, caregivers, and visitors (4.3%) were infected. however, trying to correlate patient #1's transmission capacity or epidemiological characteristics to the infection rate had limitations because with the spread of mers, the scope of surveillance of contacted individuals gradually expanded, and the standards were not consistent across different institutions. nonetheless, based on a study analyzing epidemiological characteristics of the mers outbreak around pyeongtaek st. mary's hospital, the rate of infection was 3.9% overall and was 1.1% among medical staff members; 7.6% among patients; and 3.3% among guardians, caregivers, and visitors [4] . aside from patient #1, 3 of 28 individuals, in particular, with a confirmed diagnosis (#14, #15, and #16) individually infected more than 5 individuals whose diagnosis was additionally confirmed, and they were thus labeled as "super spreaders." it is thought that, after contact with patient #1, they visited different medical facilities and became epidemiologically associated with additional patients with a confirmed diagnosis [5] . the 2015 mers outbreak in korea started with the infection of a businessman in his 60s who often traveled to the middle east for business. he did not show symptoms at his arrival in korea. a week later, he started experiencing symptoms and visited medical facilities. however, it took 9 days for the mers infection to be confirmed at the 4th medical facility he visited, and, by then, the infection transmission had already begun at the previous 3 medical facilities. thereafter, a "mers crisis" transpired for the first time wherein a total of 186 patients were confirmed with mers infection; 36 deaths occurred among these patients, and 16,693 individuals were quarantined for prevention. the clinical symptoms in the first patient deteriorated because of a delay in receiving appropriate treatment, and he was assisted with mechanical ventilation. on day 42 after admission to the 4th hospital, tests for mers virus were negative, and the patient's condition recovered. from the epidemiological investigation, the course of transmission to this patient in the middle east was not clear. it can only be inferred that the first patient became infected while visiting saudi arabia, on the basis of a recent study showing that the mers virus that broke out in korea is genetically closest to the qatar strain [5, 6] . however, to uncover the specific course of transmission, we must perform an epidemiological investigation in coordination with saudi arabia. various factors are considered as causes of the widespread outbreak. the first is health care workers' lack of knowledge regarding mers. since june 2013, the kcdc had organized a national mers management team in preparation of a mers outbreak in korea, and mockup training had been performed to enhance the capacity to manage outbreaks of novel infectious diseases at quarantine stations and at the level of local government of cities and provinces. however, in cases such as the present case, wherein the symptoms do not manifest themselves when patients enter korea, preventing an outbreak of a novel infectious disease by imposing quarantine is not practical. in particular, health care workers did not receive sufficient education or communication against mers in anticipation of a case wherein a mers-infected individual, on whom quarantine was not imposed, would go to a regional medical facility. until the diagnosis was confirmed for patient #1, the first 3 medical facilities that he visited were unable to suspect a mers case. the doctor at the 3rd medical facility, who examined patient #1 and subsequently became infected, in an interview with the media after his discharge stated that he had not even heard of mers. the second factor communication about mers among travelers to the middle east was insufficient. since 2013, when the likelihood of spread of mers would spread within the middle east region was indicated, the kcdc persistently promoted mers awareness among the travelers to that region. however, patient #1, who frequently visited the region and his family members, had no idea about mers. thus, when he visited the general medical facilities with respiratory symptoms, he did not mention about his travel to the middle east, and only mentioned about his visit to bahrain when inquired about a travel history involving the middle east at the medical center where his diagnosis was confirmed. therefore, mers could not be diagnosed sooner. a third factor contributing to the rationale behind the outbreak was not kept in check earlier was that quarantine was not thoroughly imposed. after patient #1's diagnosis was confirmed, thorough quarantine was neither imposed on individuals who had close contact with him nor on those with casual contact (such as those who had a possibly contacted the doctors or the patients or those who were exposed to a space infected by him). lastly, in this outbreak, most patients were infected via hospital-acquired infection because korean medical institutions' patient rooms and emergency rooms were very crowded, and infected patients had to visit several hospitals because of the shortcomings of the medical care delivery system, which played a critical role in the spread of mers from hospital to hospital. through an investigation on the mers outbreak within korea, valuable lessons were learned. they include (1) accumulation of epidemiological knowledge of the pattern of mers transmission as well as medical knowledge on its clinical courses; (2) improvement of epidemiological investigative techniques using cctv, global positioning system tracking, and review of health insurance review and assessment service records, among other things; (3) problems revealed in the existing preventive techniques, including the early determination of the scope of contacted people; (4) accumulation of experiences with the preventive techniques used for the first time in korea, such as cohort quarantine; (5) reconsideration of the management systems that deal with an outbreak of infectious diseases across the country, such as in the present case, at the levels of central government, local government, and the public; and (6) reconsideration of infectious disease management system at hospitals, culture involving patient visitation, and emergency room environments. the 2015 mers outbreak in korea began with an infected businessman in his 60s who visited the middle east region. however, the spread of outbreak is attributed to insufficient preparation and management by the ministry of health and welfare. to prepare for an outbreak of novel infectious diseases in future, government organizations responsible for infectious diseases need to train personnel specialized in the area, and also promote awareness among medical staff at the primary and secondary medical facilities as well as among the public. the mers outbreak should serve as an opportunity to improve the level of managing hospital-acquired infection in korea. mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome current epidemiological situation of middle east respiratory syndrome coronavirus clusters and implications for public health response in south korea epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control the authors have no conflicts of interest to declare for this study. supplementary material (korean version) is available at http: //www.e-epih.org/. key: cord-252883-1ub01j2x authors: bleibtreu, a.; bertine, m.; bertin, c.; houhou-fidouh, n.; visseaux, b. title: focus on middle east respiratory syndrome coronavirus (mers-cov) date: 2019-11-11 journal: med mal infect doi: 10.1016/j.medmal.2019.10.004 sha: doc_id: 252883 cord_uid: 1ub01j2x since the first case of human infection by the middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia in june 2012, more than 2260 cases of confirmed mers-cov infection and 803 related deaths have been reported since the 16th of october 2018. the vast majority of these cases (71%) were reported in saudi arabia but the epidemic has now spread to 27 countries and has not ceased 6 years later, unlike sars-cov that disappeared a little less than 2 years after emerging. due to the high fatality rate observed in mers-cov infected patients (36%), much effort has been put into understanding the origin and pathophysiology of this novel coronavirus to prevent it from becoming endemic in humans. this review focuses in particular on the origin, epidemiology and clinical manifestations of mers-cov, as well as the diagnosis and treatment of infected patients. the experience gained over recent years on how to manage the different risks related to this kind of epidemic will be key to being prepared for future outbreaks of communicable disease. the first case of infection attributed to middle east respiratory syndrome coronavirus (mers-cov) was detected in saudi arabia in june 2012 [1] . mers-cov then spread to several neighboring countries, mainly jordan and qatar (see fig. 2 ), and imported cases of the disease were reported throughout the world in asia, africa, europe and the americas [2] . by the 16th of october 2018, 2260 con the first two coronaviruses demonstrated to cause respiratory infections in humans, the coronaviruses 229e and oc43, were identified in the 1960s. they were held responsible for respiratory infections of moderate severity in humans. despite these viruses being identified in several reports as causing lower respiratory tract infections, it was generally accepted that coronaviruses were of low pathogenicity until the emergence of sars-cov (severe acute respiratory syndrome coronavirus) in 2002, a virus with a fatality rate estimated at 10%. the sars outbreak that resulted in more than 8400 cases was finally contained two years later, in 2004, and the virus has not been detected again since [4] . there was renewed interest in coronavirus research following the sars epidemic, and two novel endemic human coronaviruses were identified, nl63 and hku1 respectively in 2004 and 2005, but could not be replicated in cell culture. both of these new viruses were responsible for respira-tory infections of moderate seriousness like the coronaviruses 229e and oc43. great effort has been made to identify coronaviruses in animal populations, both before and after the sars outbreak, in order to better understand and control the risk of animal-to-human transmission. this resulted in the discovery of coronaviruses in numerous animal species, with a few exceptions such as sheep and goats, fish and non-human primates [5] . the first case of mers-cov infection was reported in jeddah, saudi arabia, in june 2012 [1] . the patient, a 60-year-old man, died from lung and kidney failure 11 days after being admitted to hospital. very shortly afterwards, in september 2012, a second patient was admitted to hospital in the united kingdom for severe respiratory infection related to a novel coronavirus following travel to the middle east. the new virus was found to replicate in a tissue culture model and was rapidly isolated and identified for both cases [6, 7] . retrospectively, other cases of the disease were found to have occurred before the 2 aforementioned cases: in april 2012, an outbreak at zarqa hospital in jordan affected the staff of the intensive care unit, with two fatal cases. the respiratory samples collected were later confirmed to be positive for mers-cov [8] . these initial cases were rapidly followed by a series of outbreaks in all saudi arabian provinces that were characterized by the infection of health professionals in direct contact with the patients. other similar outbreaks were observed in several neighboring countries: qatar, bahrain, kuwait, jordan and tunisia. health authorities reacted quickly to the reports of these epidemics and the strong resemblance with observed clinical features of sars-cov infections. indeed, although a few patients developed mild infections, the fatality rate for patients infected with mers-cov was over 30% [2] . following the identification of mers-cov, great effort was put into finding which animal species it originated from in order to stop the further spread of the disease to humans. mers-cov was very rapidly determined to be genotypically closely related to the betacoronavirus lineage c viruses identified in bats [9] . based on these findings, and the major role of bats in the genetic diversity and spread of coronaviruses, much of the initial work aiming at finding the natural reservoir of mers-cov focused on bats. however, no conclusive evidence demonstrating that bats were the natural reservoir of mers-cov in the arabian peninsula were found, despite the identification of closely related viruses in bats in sub-saharan africa [10] , far from the existing outbreaks. very strong epidemiological links were identified between the human cases and camels and resulted in the isolation in camels of viruses that were directly related to mers-cov and that could replicate in cultured human cells [11] . the investigation of dromedary camel serum collections, some of which collected as early as 1983, demonstrated that the virus was already widespread (seropositivity rate > 80%) in the east african countries (somalia, sudan and egypt). these countries export dromedary camels to arabian countries, but also in kenya, nigeria, tunisia, ethiopia, burkina faso and morocco [12] [13] [14] . phylogenetic analysis revealed 5 distinct coronavirus lineages in dromedary camels, including one recombinant lineage that led to the mers-cov epidemic in humans [15] . mers-cov is a betacoronavirus belonging to lineage c. it is an enveloped virus with a positive-sense single-stranded rna genome of about 30 kb. under electron microscopy, virions are generally spherical with surface projections (spikes) formed by the surface protein s creating an image reminiscent of a crown or solar corona. the positive-sense single-stranded rna genome acts as messenger rna (mrna) with a 5 cap and a 3 polyadenylated tail. it plays three roles during the host cell cycle: (i) it acts as the initial rna molecule for the infection cycle; (ii) it is the template for replication and transcription; (iii) it is the substrate that is packaged into the newly assembled viral particles [16] . the mers-cov genome is organized in the same way as other coronavirus species. the first two thirds of the mers-cov genome contain two overlapping reading frames (orf1a and orf1b) that translate into the replication-transcription complex including 16 non-structural proteins. the remaining third of the genome encodes the four structural proteins, the spike (s), envelop (e), membrane (m) and nucleocapsid (n) proteins, as well as five accessory proteins (orf3, orf4a, orf4b, orf5 and orf8b) that are not required for genome replication but are probably involved in virulence. the flanking sequences, on both ends of the genome, contain untranslated 5 and 3 regions (utr) (fig. 3 ) [17] . the viral particle can enter the cell in two ways, which probably contribute to the broad tissue tropism of this virus that replicates mainly in respiratory epithelial cells but can also infect many other cell types. via the endosomal pathway, the s1 domain of the mers-cov spike protein (s) binds its receptor, dipeptidyl peptidase 4 (dpp4) [18] , induces endocytosis of the viral particle and a change in the conformation of the s2 subunit of the s protein that then mediates virus-host membrane fusion and uncoating of virus rna. mers-cov can also enter host cells via a non-endosomal mechanism by direct fusion of the virus with the plasma membrane following s protein cleavage by human proteases [19] . following entry into the cytoplasm and uncoating of the virus nucleocapsid, the viral genomic rna is translated to produce two polypeptides, pp1a and pp1b, that form the replicase-transcriptase complex. this initial replicase-transcriptase complex uses the genomic rna to produce 16 non-structural proteins that assemble into the replication complex. the replication complex then replicates the genomic rna and produces other subgenomic rnas that ensure the translation of the structural proteins. virions are assembled at the endoplasmic reticulum membrane as viral proteins and genomic rna are grouped together and then bud into the lumen of the endoplasmic reticulum. the virions are then exported via the secretory pathway of the endoplasmic reticulum into the golgi intermediate compartment and then into the extracellular environment. the m protein drives the packaging process by selecting and organizing the viral envelop components at the assembly sites and interacting with the nucleocapsid to allow budding [20] . several large serology studies suggest that cases of asymptomatic or mild mers-cov infection occur regularly, although infrequently. the importance of such cases is difficult to assess [10] . it is therefore difficult to determine whether these cases are due to or take part in human-to-human transmission. several studies suggest that less than 50% of infected patients transmit the virus to individuals they come into contact with, even at the beginning of an outbreak [10] . the disease therefore seems to spread due to frequent animal-to-human transmission, from camels to humans, with limited subsequent human-to-human transmissions [21] . there are unfortunately exceptions to this observation and local outbreaks caused by human-to-human transmission have been observed on a regular basis, mostly in hospitals. to date, the most poignant example is the outbreak that occurred in south korea in which the index case caused 185 secondary cases, among whom 30 were care providers, leading to 24 fatalities [3] . this outbreak was characterized by the key role of a few "super spreaders", delayed diagnosis, high doctor shopping behavior and the importance of confined spaces (waiting room, hospital room, ambulance). in this example, the resemblance with sars-cov's spreading mechanisms is striking, despite lower degrees of transmission to care providers for mers-cov [22] . these regular cases of imported-mers, the most recent was reported in england in august 2018 [23] , represent a real threat of local epidemics outside saudi arabia and special screening and isolation procedures need to be implemented in units likely to receive patients suspected of mers-cov infections. when possible, the first measure to be taken is to delay departure, in particular for individuals over 65 or with chronic disease, and for pregnant women or children. such measures are nevertheless challenging to maintain today as that the virus is still present 6 years after its apparition. all other preventive measures aim at preventing both animalto-human transmission and human-to-human transmission. it is therefore recommended to avoid any contact with domestic animals (firstly dromedary camels), their secretions, raw milk and insufficiently cooked meat. it is also advised to avoid eating fruit and vegetables that might have been in contact with animal secretions if not washed and peeled by oneself. to avoid human-to-human transmission, the usual recommendations for preventing the spread of any respiratory virus should be applied: hand washing with soapy water or an alcohol-based solution, covering one's nose and mouth when sneezing, refraining from shaking hands and touching one's mouth and nose with one's hands, avoiding contact with people with respiratory symptoms. finally, a last series of recommendations focus on how to behave in case of suspicious symptoms: (i) consult a doctor as soon as symptoms occur during travel and delay the return until symptoms disappear; (ii) if symptoms occur with 14 days of returning home, consult a doctor and tell him/her about the recent travel [24] . pcr-based detection methods are currently the preferred option for detecting the virus in respiratory samples and making a diagnosis of mers-cov infection. serology tests can also be performed and are often used for second-line diagnostic investigation in patients with a high suspicion of mers-cov but negative results by direct pcr testing. various respiratory matrices can be used: nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal), and even in some cases, induced sputum. the deepest samples, tracheal aspirates and bals, show the greatest sensitivity and significantly higher viral loads [25] . the genome amplification and detection methods used (pcr) were initially mostly developed in situ and performed in biosafety level 3 (bsl-3) reference facilities. the time to results is generally relatively long, 24-48 h, due to the usual time required for conventional pcr testing to which must be added the additional preparation and sample neutralization time needed to protect the laboratory staff against this virus. the pcr methods used are generally semi-quantitative and some studies suggest a correlation between the amount of virus detected and the severity of the symptoms [26] . nevertheless, no consensus has been reached yet regarding a threshold level that could actually predicts clinical severity. targeting the envelop gene upe is recommended with confirmatory testing for orf 1a or 1b or the n gene. if results diverge, sequencing is sometimes required to obtain conclusive results [27] . today, an increasing number of commercial tests are becoming available (altona diagnostics, fast track diagnostics, primerdesing ltd.) some even with a time to results of less than 1 hour (biofire-biomérieux). some of these tests are point-of-care, or can be performed in bsl3 facilities or a standard laboratory following sample neutralization in a bsl3 facility. these commercial tests must always be validated before use to check their sensitivity and compare their performance with reference methods. as with any other acute viral infection, antibodies can only generally be detected about 10 days after the onset of symptoms. in some patients, especially those with severe infections, the time interval to antibody detection may be even longer [28] . serological testing is therefore of little help for the initial diagnosis of symptomatic patients, but can be useful for epidemiological investigations. the highly immunogenic s and n viral proteins are widely used targets for serological tests and are found on all coronaviruses. various approaches have been developed: serum neutralization assays [29] , microarrays [30] , or more recently elisa confirmed by a microneutralization test [31] . all methods are technically complex and require a high level of expertise that restrict their use to a few highly specialized facilities. the first cases of infection with mers-cov were reported in 2012 [1] . hospital-acquired mers-cov infections have been described worldwide and represented a third of all cases reported in saudi arabia in the early stages of the epidemic [1, 32, 33] . clustered hospital-acquired infections were frequently observed during the first outbreaks and probably contributed to spreading the disease from the primary site of virus infection to the whole arabian peninsula, the most striking example of hospital-acquired outbreak being the korean outbreak in 2015 [34] . care providers are often affected and represent 15-22% of cases [33] [34] [35] [36] [37] [38] [39] . most of the cases are described in middle east countries, in particular saudi arabia (73%), with a predominance of male patients (66-69% in various studies) and a mean patient age ranging from 40 to 55 years [34, 38, 40] . comorbidities are found in 46-68.6% of patients, in particular diabetes and high blood pressure, followed by other heart conditions and finally obesity [34, 37, 38, 41] . the mean incubation time is 5 to 6.5 days. the generation interval (time between the onset of symptoms of the first case and those of the second case) is 7.6 days, which is identical to that of the respiratory syncytial virus (rsv) but threefold more than the influenza virus [36, [42] [43] [44] . the main challenge of mers-cov infection is the absence of specific clinical features for differential diagnosis with other viral respiratory diseases [37, 45] . this difficulty, combined with precautionary action taken to avoid potential secondary contamination with mers-cov [46] , can result in medical confusion and inappropriate patient management due to prolonged, difficult isolation that makes it impossible to perform the necessary complementary tests while waiting for pcr results [47] . the clinical features of mers-cov infection are extremely variable, ranging from an absence of symptoms (14-80% of cases) to a flu-like syndrome, pneumonia and acute respiratory distress syndrome (ards) [37, 48] . the three most frequent symptoms are: fever (77% [iqr: 59-82]), cough (90% [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] ), and dyspnea (68% ). many other secondary symptoms have been reported, such as sputum production (40%), odynophagia (39%), digestive system signs (20%), hemoptysis (4.3%), myalgia (43%) and headache (20%) [34, 37, 41, 42] . diarrhea is significantly more frequent in patients infected with mers-cov than in patients with another acute, febrile respiratory conditions [45] . severe mers is characterized predominantly by ards, acute kidney failure, and in the most severe cases, by multiple organ failure that can be fatal [49, 50] . one third of patients develop pneumonia and 20% develop ards [51] . the median time to respiratory failure is 12 days after the onset of symptoms. depending on studies, 53 to 89% of hospitalized patients are admitted to an intensive care unit (icu) [43, 52] . since the first mers outbreak, who had documented, in october 2018, 2260 cases of mers-cov infection confirmed by laboratory testing and 803 related deaths in 27 different countries. the retrospective fatality rate varies between outbreaks, ranging from 36.5 to 60% [33, 35, 37, 38, 42] . the mortality rate of 20.4% observed for the korean outbreak is probably the most reliable epidemiologically due to the comprehensive investigations carried out [34] . the death rate is highest among patients admitted to an icu, ranging from 58% to 90% [49, 53] . in the only cohort study performed in saudi arabia, the fatality rate for mers-cov patients was of only 10% (8/80). however, the patients of this cohort were younger, had less symptoms, showed less radiological features and only 17% were admitted to an icu [37] . the findings of the latter study diverge therefore with the situations observed in other hospitals, but are perhaps a better reflection of the infection profile in the general population in which younger subjects are less symptomatic and therefore less frequently admitted to hospital. the time interval between the onset of symptoms and death ranges from 11.5 to 27 days [34, 44, 54] . finally, co-infection with other respiratory viruses, in particular influenza, has been described although the impact of such combined infections have not been evaluated [44, 55] . co-infections with bacteria have also been reported in the patients developing the most severe disease [49, 51] . there are no specific laboratory findings related to mers-cov infection. nevertheless in patients with acute respiratory infection in mers-endemic areas, mers-cov infections have been associated with normal leukocyte and/or polymorphonuclear neutrophil counts but elevated transaminases [37, 45] . moreover, hyperleukocytosis, lymphocytopenia, thrombocytopenia, hypoalbuminemia, elevated serum creatinine, ldh and crp levels, and hypoxemia (pao2/fio2 < 300) have been repeated reported in mers-cov infected patients and are associated with severity and death [34, 45] . imaging (chest x-ray and sometimes chest ct) has revealed infection-related features in 51-75% of cases. the lesions observed are uni-or multi-focal ground glass opacifications, of subpleural and lower lobe predominance, with sometimes bilateral bi-basal involvement or features of organizing pneumonia [34, 37, 42, 45] . mortality is highest in elderly, male patients with comorbidities, especially diabetes [38, 45, 56] . patients from saudi arabia and the middle east have an increased mortality rate compared with patients from korea or other countries [38, 40] . in contrast, being a medical professional significantly reduces the risk of mortality [38, 45] . other factors associated with a higher mortality risk have been described in various studies: digestive symptoms, prolonged delay between the onset of symptoms and admission to hospital, smoking, low blood pressure, impaired gas exchange, leukopenia, anemia, disturbance of liver or kidney function, use of mechanical ventilation and prolonged stay in the icu [42, 57] . for the korean outbreak in 2015, the independent risk factors for mortality were: age > 55 years, dyspnea, diabetes, chronic lung disease, systolic blood pressure at admission < 90 mmhg, hyperleukocytosis at admission (> 10,000/mm 3 ) and the use of mechanical ventilation [34] . positive pcr results for mers-cov in blood at diagnosis are associated with an increased risk of requiring mechanical ventilation, extracorporeal membrane oxygenation (ecmo) or to lead to death [58, 59] . the lack or delayed detection of mers antibodies (elisa igg and iga, or prnt) in the blood or airways is a poor prognostic factor [54, 60] . it should however be noted that no seroconversion is observed in asymptomatic mers-infected patients [54] . finally, the mers-cov viral loads in distal lung samples were higher among deceased patients [60] . in a study including 45 patients in a tertiary referral hospital in south korea: • the predictive factors for pneumonia in mers-cov patients were: age > 45 years, body temperature > 37.5 • c on day 3, platelet counts < 150,000/mm 3 , lymphocytopenia (< 1000/mm 3 ), crp ≥ 20 mg/l and high viral loads (ct value < 28.5); • the predictive factors for respiratory failure were male sex, high blood pressure, thrombocytopenia, lymphocytopenia, hypoalbuminemia < 35 g/l and crp ≥ 40 mg/l. the patients with at least two, one and none of the predictive pneumonia factors developed pneumonia in 100%, 50% and 0% of cases, respectively [61] . several therapeutic options targeting various viral elements are currently available or under development (fig. 4) [62] . the different classes of available treatment are (i) immunotherapy with specific anti-mers-cov antibodies, (ii) molecules with antiviral activity, (iii) symptomatic treatment. few molecules have shown real curative action and the reports in the literature generally describe isolated cases or small series of cases. more studies have focused on associated treatment and supportive care. at this time, preventive therapies are still in preclinical stages. the efficacy and safety of plasma from convalescent patients have not been assessed. three separate reports concluded that such therapeutic approaches were inappropriate [63] . one trial is listed on www.clinicaltrials.gov. two cases of therapy with intravenous polyclonal iggs have been reported. in one of them, the iggs originated from donors in regions negative for mers specific antibodies. several monoclonal antibodies were tested and seemed to show anti-mers-cov activity in vitro [64] . no clinical trials are currently underway. recently, a phase i placebo-controlled, dose escalation study evaluated the efficacy of polyclonal iggs produced by transchromosomal cattle with human immunoglobulin genes immunized with the mers-cov spike (s) protein [65] . the primary outcome of tolerance to a single dose was reached. the secondary pharmacodynamic endpoint (serum neutralization activity) showed efficacy with a dose of 50 mg/kg. no phase ii trials are currently underway. a phase i study has been registered to assess the immunogenicity and tolerance of a combination of two monoclonal anti-mers-cov antibodies. the study has not yet started recruiting patients. infection with mers-cov reduces the host's interferon response. mers-cov is 100 times more sensitive to ifn-␣. treatment with ifn-␣ has been reported for many clinical cases and several retrospective cohort studies have been performed, in combination with ribavirin, lopinavir or mycophenolate mofetil (mmf). none of these studies have demonstrated increased overall survival. one study reported increased survival at d14 but not at d30 for critically ill intubated and ventilated patients [66] . a ifn/mmf combination trial is currently underway (see below). high doses of ribavirin have shown anti-mers-cov activity in vitro. ribavirin has been used to treat patients in saudi arabia as well as in france for the most severe cases managed in icus [67] . no significant effects were demonstrated either on the mortality rate or the time spent in the icu. ritonavir-boosted lopinavir has shown efficacy against mers-cov in vitro. as a result, the fda has extended the indications of lopinavir to patients infected with mers-cov. two case reports (in greece and korea) have described improvement in patients treated with lopinavir, type 1 interferon and ribavirin [68] . a phase ii-iii clinical trial is registered on clinicaltrials.gov. the aim of this study is to evaluate the feasibility, efficacy and safety of the combination lopinavir/ritonavir/recombinant ifn␤-1b vs. a placebo in patients with confirmed mers receiving optimal symptomatic care. chloroquine is among the molecules approved by the fda following in vitro studies. no clinical data or studies support its use in vivo at the present time. in vitro, anti-mers-cov activity has been demonstrated for doses of nitazoxamide that could be reached with two daily oral doses. no clinical data or studies support its use in vivo at the present time [69] . in vitro, anti-mers-cov activity has been demonstrated for doses of mmf that are acceptable for use in humans. mmf seems to show a synergistic effect with ifn-␤1b in vitro [70] . but in a non human primate common marmosets model, animals treated with mmf developed more severe lesions and showed a higher case fatality rate compared with untreated animals [70] . in contrast with animal model, the combination ifn-␤1b/mmf was administered to 8 patients in saudi arabia. all the patients survived but had lower apache ii scores that other patient groups [71] . alisporivir has been shown to provide additive in vitro anti-mers-cov activity when used in combination with ribavirin. no clinical data or studies support its use in vivo at the present time [72] . silvestrol is a molecule of the flavagline family found in plants. it binds to eif4a and enhances the affinity of eif4a for mrna. this blocks helicase activity and inhibits protein translation. a recent in vitro study demonstrated that silvestrol has anti-mers-cov activity [73] . no clinical data or studies support its use in vivo at the present time. corticosteroid therapy is currently the most widely studied therapeutic option. in a retrospective study, arabi et al. [67] compared the outcome of 309 patients with confirmed mers-cov infection managed in an icu setting and treated with (151) or without (159) corticosteroid therapy. the overall fatality rate was 67%. univariate analysis showed that mortality in the icu, during the hospital stay or at 90 days was higher in the corticosteroid group. then, following adjustment using a marginal structural model for causal inference, corticosteroid therapy was shown not to be associated with mortality, but delayed virus clearance. these findings, together with the absence of any description of the adverse effects caused by corticosteroid treatment, argue against the use of corticosteroids. a retrospective study was recently carried out in saudi arabia in mers-cov patients with refractory respiratory failure [74] . the patients were included in the study from 2014 to 2015 in five icus. the study consisted of two patient groups: ecmo versus conventional treatment. the primary endpoint was inhospital mortality. secondary endpoints included the length of stay in the icu and in hospital. thirty-five patients were included: 17 were treated with ecmo and 18 received conventional care. both groups had similar baseline characteristics. inhospital mortality was lower in the ecmo group (65 vs. 100%; p = 0.02) although they stayed longer in the icu (median stay of 25 days vs. 8 days; p < 0.01). the overall time in hospital was similar in both groups (median stay of 41 vs. 31 days; p = 0.421). in addition, patients in the ecmo group showed improved pao2/fio2 values at 7 and 14 days after admission into the icu (124 vs. 63, and 138 vs. 36, respectively; p < 0.05), and lower levels of vasoactive amines at d1 and d14 (29 vs. 80%, and 36 vs. 93%, respectively; p < 0.05). the results of this study support the use of ecmo as salvage treatment for mers patients with respiratory failure, as is the case for other respiratory infections. two trials with candidate vaccines are currently registered at https://clinicaltrials.gov/ct2/home. a phase-i clinical trial on healthy volunteers was set up to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls-5300) that expresses the s protein of mers-cov. this trial was planned to last one year and started in 2016. no results are available yet. a second phase-i trial was started by oxford university in january 2018. it uses a chimpanzee adenovirus vector containing the mers-cov s protein gene [75] . patient inclusion is currently underway. many other candidate vaccines using various different technologies are at a less advanced stage of development. the mers epidemic started in 2012. in contrast with sars-cov that disappeared 2 years after it first appeared, mers-cov continues to persist in the middle east 6 years later. although the disease has not become pandemic, outbreaks have occurred worldwide. today, it is impossible to predict with certainty whether mers-cov will disappear or continue to remain a threat for human populations. efficient vaccine development for host ani-mals and humans could play a key role in tilting the balance from potentially-pandemic to mers-cov elimination. furthermore, the epidemiological and viral determinants of the emergence of mers-cov in the middle east are difficult to comprehend, due to the high seropositivity rate of african dromedary camels but no similar disease in local human populations. the constant increase of transcontinental travel, in particular towards the main focal points of mers outbreaks with religious pilgrimages and mass tourism, raises the problem of the management of patients suspected of mers-cov infection and the absence of efficient treatment options to this date. the main problem in non-epidemic countries is to detect a mers-cov case among a great number of non-mers patients. in france, with the exception of the first 2 cases, no further cases have been detected. the current strategy is to isolate any suspicious cases as rapidly as possible to contain the infection and prevent local outbreaks as seen in south korea. the ability to rapidly test patients suspected to have mers-cov infection is the cornerstone of this strategy. the experience gained over the last few years by the health community will also help deal with any respiratory infections that will emerge in the future. the authors declare that they have no competing interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov). who mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) molecular evolution of human coronavirus genomes genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans severe respiratory illness caused by a novel coronavirus who | 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daejeon, the republic of korea ifn-(2a or ifn-(1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients prevention and treatment of respiratory viral infections: presentations on antivirals, traditional therapies and host-directed interventions at the 5th isirv antiviral group conference feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study a review of treatment modalities for middle east respiratory syndrome corticosteroid therapy for critically ill patients with the middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-(1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model broad-spectrum antiviral activity of the eif4a inhibitor silvestrol against corona-and picornaviruses extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice the chapters on the origin, emergence, structure, transmission mechanisms, prevention and diagnostic methods were mainly written mb, nh, and bv.bv produced the figures. the chapters on clinical presentation, prognosis and available treatment options were mainly written by ab and cb.all authors read, amended and agreed with the entire final manuscript. key: cord-293505-1t3hg4wi authors: bernard-stoecklin, sibylle; nikolay, birgit; assiri, abdullah; bin saeed, abdul aziz; ben embarek, peter karim; el bushra, hassan; ki, moran; malik, mamunur rahman; fontanet, arnaud; cauchemez, simon; van kerkhove, maria d. title: comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from 2015 to 2017 date: 2019-05-14 journal: sci rep doi: 10.1038/s41598-019-43586-9 sha: doc_id: 293505 cord_uid: 1t3hg4wi since its emergence in 2012, 2,260 cases and 803 deaths due to middle east respiratory syndrome coronavirus (mers-cov) have been reported to the world health organization. most cases were due to transmission in healthcare settings, sometimes causing large outbreaks. we analyzed epidemiologic and clinical data of laboratory-confirmed mers-cov cases from eleven healthcare-associated outbreaks in the kingdom of saudi arabia and the republic of korea between 2015–2017. we quantified key epidemiological differences between outbreaks. twenty-five percent (n = 105/422) of mers cases who acquired infection in a hospital setting were healthcare personnel. in multivariate analyses, age ≥65 (or 4.8, 95%ci: 2.6–8.7) and the presence of underlying comorbidities (or: 2.7, 95% ci: 1.3–5.7) were associated with increased mortality whereas working as healthcare personnel was protective (or 0.07, 95% ci: 0.01–0.34). at the start of these outbreaks, the reproduction number ranged from 1.0 to 5.7; it dropped below 1 within 2 to 6 weeks. this study provides a comprehensive characterization of mers hca-outbreaks. our results highlight heterogeneities in the epidemiological profile of healthcare-associated outbreaks. the limitations of our study stress the urgent need for standardized data collection for high-threat respiratory pathogens, such as mers-cov. such large healthcare-associated (hca) outbreaks have mainly been limited to the kingdom of saudi arabia (ksa) and the united arabian emirates (uae) until the spring 2015, when a single imported case of mers returning from the middle east initiated a cluster of 186 cases in the republic of korea (rok) across at least 17 hospitals and much of the country 18 . super spreading events in healthcare settings has been described for several previous mers outbreaks, including an outbreak in al-hasa governorate in 2013 and during the outbreak in rok, where approximately 80% of the transmission events were epidemiologically linked to five mers cases 14, 18, 23 . superspreading events in health care facilities have been observed in similar high threat respiratory disease pathogens, such as severe acute respiratory syndrome (sars) in canada, china, singapore [24] [25] [26] . while more than half of the laboratory confirmed mers-cov infections reported globally to date are associated with human-to-human transmission in healthcare settings 27 , there has been little human-to-human transmission reported in household settings 28 . outbreak investigations and scientific studies conducted during or after mers hospital outbreaks have identified that aerosol-generating medical procedures with improper or inadequate personal protective equipment place medical personnel and patients sharing wards with mers patients and family visitors at higher risk for mers-cov infection 29, 30 , with exposure to infectious droplets being the likely source of contamination. although close unprotected contact with a mers patient is generally considered necessary for human-to-human transmission 31 , several studies have revealed that mers-cov particles can persist on surfaces as long as several days, raising the possibility of a role of fomites in transmission 32, 33 . fomite transmission is further supported by observed viral spreading between rooms that were clearly separated 15, 18 and outbreaks that occurred in hemodialysis units 14, 15 . factors leading to healthcare-associated outbreaks include overcrowding in emergency departments, slow triage and isolation of suspected patients and inadequate compliance to infection prevention and control procedures 17, 23, 34 . however, few studies have described or compared the characteristics of hca-outbreaks as a whole in terms of their size, epidemiologic factors 34, 35 , or the role of interventions to stop transmission 23, 36 . here, we provide the largest comprehensive study of eleven healthcare-associated outbreaks that occurred between 2015 and june 2017. we carried out a comparative analysis of these outbreaks in terms of epidemiological profiles, demographic characteristics and clinical outcome. study design. we analyzed epidemiological datasets of laboratory-confirmed mers patients and focused our study on eleven healthcare-associated outbreaks that were reported in ksa and rok since 2015, when policies and procedures for case identification and comprehensive contact identification and follow up became systematic and were implemented by affected countries. the data used documented mers-cov infections reported to who under the international health regulations (2005). we only included clusters of cases/outbreaks that were linked to healthcare facilities. supplemental rok case-based data were provided as a detailed line list of the korean mers cases included in a published study 17 . we defined laboratory-confirmed mers-cov infection as following who guidelines 4, 37 . we defined a hca-outbreak as the occurrence of 5 or more laboratory-confirmed mers-cov infections with reported epidemiologic links between cases and during which the human-to-human transmission events were documented within a single healthcare facility, with no more than 14 days apart between cases symptom onset. the mers outbreak in the republic of korea in 2015 is treated as a single outbreak. individual-level variables included information on age, sex, nationality, occupation (healthcare personnel (hcp) yes/no), dates of symptom onset, date of notification to who, presence of any pre-existing co-morbid conditions, and clinical outcome. in case of missing or conflicting information and when information from the country was not available, we considered the data as missing. statistical analysis. descriptive analysis was performed by hca-outbreak (outbreak-level analysis) using aggregated data, and for all cases (individual-level analysis). all analyses were conducted using stata, version 14 (college station, tx: statacorp lp), microsoft excel (version 15.35 2017, jones, chicago usa) and r. outbreak-level analysis. we calculated the duration, size and case fatality ratio for each outbreak. the duration of an outbreak was calculated as the number of days between the date of symptom onset of the first reported case to the date of symptom onset of the last reported case. we obtained weekly smoothed estimates of the case reproduction number based on the approach developed by wallinga and teunis 38,39 using the r 0 package. we assumed that the serial interval of mers-cov had a gamma distribution with a mean of 6.8 days and a standard deviation of 4.1 days, as described elsewhere 40 . individual-level analysis. we summarized case characteristics as frequencies and proportions for categorical variables, as median and interquartile ranges (iqr) for continuous variables. chi-square tests were used to compare subgroups of cases when appropriate. a p value of less than 0.05 was used to indicate statistical significance. univariate analysis identified variables significantly associated with fatal outcome, which were included in a multivariable model. model selection was performed using a multilevel mixed-effects logistic regression with backwards selection taking into account clustering of individuals by outbreak. for the variable "age", the cut-off was fixed at 65, based on the results of the univariate analysis. variables with p-values < 0.05 were retained in the final model. all data used in these secondary analyses were de-identified data obtained from who or datasets from peer-reviewed literature. as such, these data were deemed exempt from institutional review board assessment. (2019) 9:7385 | https://doi.org/10.1038/s41598-019-43586-9 www.nature.com/scientificreports www.nature.com/scientificreports/ general characteristics of hca-outbreaks. since 1 january 2015 to 1 october 2018, 2,260 laboratory-confirmed mers-cov infections have been reported to who. figure 1a illustrates the global epidemic curve since mers was first identified in humans. each peak is associated with a health care associated outbreak (colored lines, fig. 1a ). from 2015, affected countries implemented systematic contact tracing and follow up (including laboratory testing), investigation and data collection of mers suspect cases 41, 42 . in our analysis, a total of 423 laboratory-confirmed mers cases from eleven distinct hca-outbreaks during 2015-2017 were included ( table 1 ). the eleven hca-outbreaks varied in terms of duration, size and epidemiological profile ( table 1, fig. 1b) . the median number of total reported cases per outbreak was 10 (interquartile range, [iqr] 6-27), ranging from 5 to 186 cases. the median duration was 20 days (iqr 16-23), ranging from 10 to 57 days. three outbreaks began with sporadic cases during the first two to five weeks of the outbreak, while the other eight displayed a rapid increase to the peak. the median time between onset of symptoms of the first reported case and the peak of incidence was 3 weeks (iqr 2-3.75), ranging from 2 to 6 weeks. the case fatality ratio (cfr) in outbreaks was 28% (116 reported deaths among 423 cases), compared with the global overall cfr of 35.5% (800 reported deaths among 2,254 cases reported as of 1 october 3 (table 1) . during hca outbreaks, cfr ranged from 0 to 75% (p < 0.01) and cfr was significantly lower among hcp mers-cov infections compared to non-hcp mers-cov infections (2% vs. 36% p < 0.01). demographic and clinical characteristics. the demographic and clinical characteristics of the cases from hca outbreaks included in our analyses are summarized in table 1 . the median age was 54 (iqr, 36-65), and significantly varied by outbreak (p < 0.001). five outbreaks had a median age <40 and the other 6 outbreaks had a median age ≥50. the majority of cases were male (57%, n = 243/423), and the sex ratio among cases differed significantly between outbreaks (p < 0.001). the overall proportion of hcp was 25% (n = 105/422). this proportion varied significantly by outbreak (p < 0.001), from 13% to 89% (table 1) . median age was significantly lower among hcp than non-hcp cases (35 iqr 29-46 vs 58 iqr 45-70, p < 0.001) and the proportion of females was higher among hcp than non-hcp (70% vs 33%, n = 422, p < 0.001). more than half (57%, n = 214/377) of cases had at least one underlying co-morbid condition (table 1) , and this was significantly lower among females compared to males (46% vs 64%, respectively, n = 377, p < 0.001) and among hcp compared to non-hcp (13% vs 70%, n = 376, p < 0.001). sixteen percent (n = 67/419) of cases were asymptomatic at time of reporting (table 1 ). this proportion varied significantly between outbreaks ranging from 0% to 87% (n = 419, p < 0.001, fig. 1c ). median age of asymptomatic cases was 34 (iqr, [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] , the majority of whom were females (70%, n = 47/67) and had no underlying co-morbid conditions (78%, n = 29/37). the proportion of hcp among asymptomatic infections was high (70%, n = 47/67), and the cfr was null. the median duration between symptom onset and case notification to who was 5 days (iqr 3-8). risk factors associated with fatal outcome. in univariate analysis, fatal outcome was significantly associated with age (p < 0.001), presence of underlying comorbidities (p < 0.001), non-hcp status (p < 0.001), and male sex (p < 0.001, estimation of time-varying reproduction number. at the start of each hca outbreaks, the case reproduction number r (t) ranged from 1.0 (95% ci 0.7-1.3) to 5.7 (95% ci 3.0-9.0) ( table 1 and fig. 2 ). estimates of r (t) dropped below 1 within 2 to 6 weeks from the first reported case in the outbreak (n = 11 outbreaks, median 3 weeks, iqr 2-4). year of outbreak period of time* www.nature.com/scientificreports www.nature.com/scientificreports/ we provide here a comparative characterization of mers hca-outbreaks and report substantial heterogeneity between hca-outbreaks illustrating the complexity of the factors contributing to the emergence of a cluster of cases associated with nosocomial transmission. the duration and epidemic profiles of outbreaks varied; some started with an apparent sharp increase in incidence while others began more slowly with isolated cases emerging intermittently for a few weeks before a cluster of cases appeared in a healthcare facility. some outbreaks had a sharp decline in cases, while others experienced a long tail lasting several weeks after the peak. the median estimates of the reproduction number r(t) in the early stages of outbreaks included in our analyses reached as high as 5.7 in the republic of korea, as has been found by others 43 , likely facilitated by multiple superspreading events at two hospitals 43 . what is perhaps most informative from a public health perspective is the length of time it took to bring the outbreaks under control. all of outbreaks reached r t values below 1 within 2 to 6 weeks after the first cases were identified, highlighting that the time frame in which hospital and ministry officials can implement control measures to stop nosocomial outbreaks. factors explaining differences in hca outbreak size and duration might include variations in the speed in which cases were suspected and timing of interventions implemented in healthcare settings, including contact identification, management and isolation of patients, improved infection prevention and control measures and in some cases, the requirement to close departments [14] [15] [16] [17] [18] 29 . in this study, we were not able to evaluate the impact of interventions in these outbreaks. prevention of large hca outbreaks since 2014 (fig. 1a) , may be, in part, explained by improvements in contact tracing policies implemented in 2015. in 2015, contact tracing became more systematic with the identification and follow up of high (close, unprotected contact) and low risk contacts (protected hcw). in affected countries, national ministries of health and hospital staff comprehensively list all contacts of known mers patients, including healthcare workers at all facilities/departments the patient visited, patients who shared wards/ rooms with mers patients, family and visitors and occupational contacts. follow up of contacts includes the testing of all high-risk contacts, regardless of the development of symptoms. recommendations stated that positive contacts are placed in quarantine (home or hospital isolation for asymptomatic or symptomatic secondary cases, respectively) until they test negative 41, [44] [45] [46] . additionally, affected countries enhanced infection prevention and control procedures education, and training, and implemented visual triage systems 41 to reduce delays in testing, isolation and care of suspected mers-cov patients. this has again been recently illustrated by the lack of secondary cases following the identification of a confirmed case of mers in korea in september 2018 47 was due to the rapid and comprehensive isolation, treatment and management of contacts of the patient. the variation in outbreak size and duration is also affected by superspreading events early in some outbreaks, during which a limited number of cases generated a disproportionately large proportion of the secondary cases under specific conditions in hospitals, occurring in some outbreaks [48] [49] [50] . two super spreading events have been documented in ksa and in the republic of korea. in the republic of korea, the practice of "doctor shopping", extended stays in overcrowded emergency departments, cultural practices of large numbers of family members visiting sick relatives, and environmental contamination amplified transmission from some patients to others 14, 17, 18, 51 . during the outbreak in ksa in 2015 at the ministry of the national guard hospital, a high number of secondary cases were among hcp very quickly after the hospitalization and a surgical procedure of the index case 16 . these events triggered comprehensive review ipc in hospitals, emergency department layout, movements of patients, triage of respiratory visits, duration of emergency department stay, training of hospital staff and disinfection of healthcare facilities. our study confirmed that age and presence of comorbidities are linked to increased risk of death, similar to previously published results 52, 53 whereas being hcp was protective. the protective effect of hcp could be explained by the fact that hcp are more likely to be younger (<60 years old) and have fewer underlying medical conditions than hospitalized patients, but also that they are likely to be identified earlier or seek medical care soon following contact with a confirmed patient. the proportion of asymptomatic secondary cases identified during outbreaks has increased since 2014. there is no evidence to suggest that this represents changes in virus pathogenicity, epidemiology or transmission www.nature.com/scientificreports www.nature.com/scientificreports/ patterns of mers in recent years. however, the increase in the number of reported asymptomatic cases is hypothesized to be due to earlier detection efforts from more aggressive contact identification and testing during hca-outbreaks since 2015 as testing policies adopted and implemented by ksa and other countries have changed following the large outbreaks in jeddah/riyadh in 2014 3, 41, 54 . in 2017, 40-80% of the laboratory confirmed hcp secondary cases experienced no symptoms and were detected as part of a policy to test all contacts irrespective of symptoms (table 1) . we believe that the identification of hcp asymptomatic cases, and their subsequent isolation, has had a strong impact on prevent further human to human transmission in health care settings. this is visually demonstrated in fig. 1c by the outbreak labelled sau16_2, which included 26 (of 30 www.nature.com/scientificreports www.nature.com/scientificreports/ reported cases) asymptomatic cases. while this is a large number of secondary cases, we argue that the early identification, isolation and recovery of these asymptomatic/mildly symptomatic cases effectively stopped human to human transmission. our study has several limitations due to variability in the completeness and quality of case-based data provided to who since 2012 and also due to the lack of detailed information on the timing specific interventions were implemented in relation to each outbreak. without detailed information on the timing of interventions in each health care facility it was not possible in our analyses to determine which intervention or combination of interventions had the greatest impact on stopping the mers outbreaks. moreover, prior to 2015, contacts without symptoms were not tested for mers-cov infection, thus the rate of identification of secondary cases was drastically different prior to 2015, which complicates the comparison of data collected before and after 2015. the improvements in data reporting on cases (e.g., more systematic reporting of underlying conditions, reported exposures, contacts between patients) from 2015 allowed us to perform better analyses with less missing values. we continue to encourage the policy of identifying, following and testing of all high risk contacts of mers patients in hca-outbreaks 3, 41, 55 . the natural history of asymptomatic infection and role of asymptomatic or mildly symptomatic hcp in transmission of the virus between patients, requires detailed scientific studies to better understand their potential role in transmission 15 . the sharing of outbreak experiences between affected hospitals within and between countries and a detailed evaluation of the impact of non-therapeutic interventions is critical to our understanding and for the prevention of nosocomial outbreaks of respiratory pathogens. health care professionals and hospitals currently have tools to limit the extent and impact of such events, which include early identification and isolation of suspect patients and strict adherence to standard infection prevention and control measures. these are the hallmark of effective mers-cov control. a combination of interventions including the efficient triage of patients with respiratory symptoms at hospital entry; limiting wait times and overcrowding in waiting areas; isolation of suspected and confirmed cases; appropriate use of droplet personal protection equipment by hcp; basic hand hygiene; increased protective aerosol precautions for hcp during aerosol-generating medical procedures; efficient surface and environmental 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respiratory syndrome (mers) cases given an imported case the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea transmission characteristics of mers and sars in the healthcare setting: a comparative study risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea middle east respiratory syndrome risks of death and severe disease in patients with middle east respiratory syndrome coronavirus asymptomatic infection of middle east respiratory syndrome coronavirus using serologic survey in korea world health organization. disease outbreak news the authors would like to thank the many individuals involved in the collection of case-based data and in the care competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-278238-w1l8h8g8 authors: okba, nisreen ma; raj, v stalin; haagmans, bart l title: middle east respiratory syndrome coronavirus vaccines: current status and novel approaches date: 2017-04-13 journal: curr opin virol doi: 10.1016/j.coviro.2017.03.007 sha: doc_id: 278238 cord_uid: w1l8h8g8 middle east respiratory syndrome coronavirus (mers-cov) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. the re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. ongoing efforts to develop mers-cov vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. extending on our current knowledge of mers, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish mers-cov transmission to humans. in addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next cov outbreak. nisreen ma okba, v stalin raj and bart l haagmans middle east respiratory syndrome coronavirus (mers-cov) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. the re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. ongoing efforts to develop mers-cov vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. extending on our current knowledge of mers, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish mers-cov transmission to humans. in addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next cov outbreak. coronaviruses are the largest positive sense single stranded rna viruses. there are six human coronaviruses (hcov) to date; hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, severe acute respiratory syndrome (sars)-cov, and middle east respiratory syndrome (mers)-cov. prior to the sars-cov epidemic in 2002-2003, covs were known to cause mild respiratory infections in humans. sars-cov, on the other hand, infected around 8000 cases causing severe respiratory disease with a 10% fatality rate [1] . ten years later, mers-cov emerged in the human population also to cause severe respiratory infection [2] . in contrast to the sars-cov epidemic, which was contained within one year, mers-cov still continues to cause outbreaks with increasing geographical distribution, four years after its first identification. as of march 2nd 2017, 1905 cases in 27 countries have been reported to the who with 677 deaths accounting for a 35% case fatality rate (http://www.who.int/emergencies/mers-cov/en/). like sars-cov, mers-cov emerged as a result of zoonotic introduction to the human population. despite its close genome similarity with bat coronavirus hku4 and hku5 [2] , accumulating serological and molecular evidence pointed to dromedary camels as the most probable reservoir for mers-cov [3] [4] [5] . this poses a continuous risk of virus spill-over to people in contact with camels, such as those working in slaughter houses and animal farms, evidenced by the presence of mers-cov antibodies in sera of those individuals [6, 7] . nosocomial transmission, however, accounts for the majority of mers-cov cases reported in outbreaks [8] [9] [10] , although a substantial part of infections that occur result in unrecognized asymptomatic or mild illnesses [11] . thus, in addition to camel contacts, other highly-at-risk groups are healthcare workers and patient household contacts [8, 12, 13] . considering the ongoing mers-cov outbreaks, it is crucial to develop intervention measures among which vaccines play an important role. despite the fact that the emergence of mers-cov and sars-cov has dramatically changed the way we view covs, there is no licensed cov vaccine or therapeutic drug available to date [14, 15] . a cornerstone for rational vaccine design is defining the determinants of immune protection. accumulating data from studies done so far on mers-cov and other coronaviruses revealed that a combination of both virusspecific humoral and cellular immune responses is required for full protection against coronaviruses. especially neutralizing antibodies are considered key players in the protective immunity against covs. neutralizing monoclonal antibodies (mabs) reduced viral loads in mers-cov receptor-transduced mice, rabbits and macaques [16] [17] [18] [19] . similarly, convalescent camel sera increased virus clearance and decreased lung pathological outcomes in mice with an efficacy directly proportional to anti-mers-cov-neutralizing antibody (nab) titers [20] . also polyclonal sera produced in transchromosomic bovines protected mice against mers-cov challenge [21] . evidence for the protective role of antibodies also comes from recent studies analyzing immune responses in patients that survived or succumbed to mers-cov. although neutralizing antibodies were only weakly inversely correlated to viral loads, serum antibody responses were higher in survivors compared to fatal cases but viral rna was not eliminated from the lungs [22, 23] . administration of convalescent sera, however, did not lead to significant reduction in viral loads [22, 24] . the presence of mucosal iga abs, on the other hand, was found to influence infectious virus isolation [25] . besides humoral immunity, cellular immune responses are also considered to play a crucial role in protection against coronaviruses. while b-cell deficient mice were able to clear mers-cov, those lacking t-cells failed to eliminate the virus, pointing out the crucial role of t-cells in viral clearance [26] . this is supported by the observation that t-cells were able to protect aged mice against sars-cov infection [27 ] and the fact that a reduced tcell count was associated with enhanced disease severity in sars patients [28] . along with other studies, these data highlight the importance of t-cells for virus clearance and protection against mers-cov [26, 27 ] and sars-cov [29, 30] . it is also noteworthy to mention that while neither antibodies nor memory b cells were detectable 6-years post-infection [31] , sars-cov-specific memory t-cells, despite being low in frequency, persisted up to 11 years post-recovery [32] . nonetheless, the protective capacity of such memory response is not known. hence, taking into account the waning of virus-specific humoral responses, generating a long-lived memory t cell response through vaccination could be favorable, but as proper b-and t-cell immune responses are required for efficient protection, vaccination should target the induction of both. at the moment we lack information concerning the longevity of anamnestic immune responses following mers-cov infection, except for a recent study showing that antibody responses, albeit reduced, persisted up to 34 months post-infection [33] . the role of immune responses in protection is also in line with the observed increased fatality among the aged population following mers-cov infection. retrospective studies on mers-cov patients from saudia arabia and south korea have found a significant correlation between old age and mortality [8, 13, [34] [35] [36] , a pattern that has been also reported for other respiratory viruses such as sars-cov [1] and influenza virus [37] . this is most likely caused by immunosenescence; a failure to produce protective immune response to new pathogens in elderly due to impaired antigen presentation, altered function of tlrs, and a reduced naïve b and t cell repertoire [38] . this agerelated increase in mortality was also reported in sars-cov laboratory-infected animals, that is, mice and nonhuman primates (nhps) [39, 40] , and was associated with low neutralizing antibodies and poor t-cell responses [41, 42, 43 ] . several factors that play a role in t-cell activation were also found to be dysregulated in an age-related manner. age-related increase in phospholipase a 2 group iid (pla 2 g2d), and prostaglan-dind 2 in the lungs contributed to a diminished t-cell response and severe lung damage through diminishing respiratory dendritic cell (dc) migration [44, 45] . likewise, adoptive transfer of t-cells to mice enhanced viral clearance and survival [29] , highlighting the contribution of a reduced t-cell response in severe disease outcome. these observations also highlight the need for more effective preventive measures for the elderly. in this sense, induction of a potent airway t-cell response may be crucial to protect against covs [27 ] . thus, a promising approach to protect against mers-covinduced fatality is to enhance virus-specific tissue (airway) resident memory t-cell responses through intranasal vaccination. although the mers-cov genome encodes for 16 nonstructural proteins (nsp1-16) and four structural proteins, the spike (s), envelope (e), membrane (m), and nucleocapsid (n) [46] , the viral structural proteins, s and n, show the highest immunogenicity [47] . while both s and n proteins can induce t-cell responses, neutralizing antibodies are almost solely directed against the s protein, with the receptor binding domain (rbd) being the major immunodominant region [48] . thus, current mers-cov vaccine candidates mainly employ the spike protein or (parts of) the gene coding for this glycoprotein. these mers-cov vaccines candidates were developed using a wide variety of platforms, including whole virus vaccines, vectored-virus vaccines, dna vaccines, (table 1) and protein-based vaccines ( table 2) . although live attenuated vaccines produce the most robust immune responses, they pose a risk from reversion to virulence. inactivated virus vaccines may cause harm due to incomplete attenuation or the capacity to induce lung immunopathology [49] . viral-vector-based vaccines, on the other hand, provide a safer alternative and have been developed using modified vaccinia virus ankara (mva) [50, 51, 52 ] , adenovirus (adv) [53, 54] , measles virus (mev) [55] , rabies virus (rabv) [56] , and venezuelan equine encephalitis replicons (vrp) [26, 57] , all expressing mers-s/s1 proteins. additionally, vrp expressing the n protein have also been developed [27 ] . a major hurdle facing these viral-vector-based platforms is preexisting immunity in the host which potentially can impair the vaccine efficacy. however, this can be prevented by using virus strains not circulating in the targeted population or immunization strategies involving heterologous prime-boost immunization, for example, mva and adv. although plasmid dna vaccines are considered to be of low immunogenicty in humans, current versions developed seem to induce potent immune responses. dna-based vaccines directed at inducing anti s responses were also shown to exert protection in nhps [58, 59] . noteworthy to mention is middle east respiratory syndrome coronavirus vaccines: current status and novel approaches okba, raj and haagmans 51 ad/hdpp4-mice, mice transduced with hdpp4 in an adenoviral vector; alum, aluminum hydroxide; e, envelope protein; hdpp4, human dipeptidyl peptidase 4; i.m., intramuscular; i.n., intranasal; m, matrix protein; mers-cov, middle east respiratory syndrome coronavirus; nab, neutralizing antibodies; nd, not done; nhp, non-human primate; rntd, recombinant n-terminal domain; rbd, receptor-binding domain; rrbd, recombinant rbd; rbd-fc, rbd fused to the antibody crystallizable fragment of human igg; s, spike protein; s1, s1 domain of spike protein; s367-606, amino acid residues 367-606 of the s protein; s736-761-klh, peptide s736-761 coupled to keyhole limpet haemocyanin; s.c., subcutaneous; vlps, virus-like particles; a i.m.;alum/cpg odn produced higher neutralizing antibody responses than s.c.; ifa/cpg odn. b s350-588-fc, s358-588-fc, s367-588-fc, s367-606-fc, and s377-588-fc were tested and s377-588-fc had the highest nab titers although some produced equal s1 igg response [66] . c mf59 produced the highest immunogenicity at low doses of antigen compared to s377-588-fc only, or with freund's/alum/mpla-sm/isa51/mf59. [67] 1 mg of antigen with mf59 was sufficient to produce humoral and cellular immune responses similar to higher doses (5 mg or 20 mg) [68] . d i.n. + poly(i:c) vaccination induced stronger systemic cellular responses and higher local immune responses in mice lungs (iga and neutralizing antibody titers) than s.c. + montanide isa51 vaccination. that a combination of dna (s) and recombinant protein (s1) in a heterologous prime-boost immunization strategy induced higher immune response (nab) compared to each component alone [58] . additionally, protein-based vaccines were developed in various platforms as virus-like particles (vlps) [60] , nanoparticles [61] , peptide-based [62] , and subunit vaccines directed against various regions of the spike protein s1 [58] , the n-terminal domain [63] , and the rbd [48, [64] [65] [66] [67] [68] [69] [70] . those vaccines have the highest safety profile among vaccine platforms but confer variable degrees of immunogenicity which need adjustment for the dose, adjuvants, and site of administration to get optimal protective responses. adjuvants influence the type and magnitude of immune response produced by vaccines, and thus the doses used [61, 65, 68] . additionally, the route of administration is a determining factor for the type of vaccine-induced immune response produced in the host. while intranasal (i.n.) vaccination with sars-n produced a protective airway t-cell response against sars-cov in mice, subcutaneous (s.c.) vaccination, inducing systemic t-cell responses, did not [27 ] . likewise, i.n. vaccination with mers-rbd induced a significantly higher neutralizing and iga antibody responses in the mice airways compared to s.c. vaccination [70] . this is important because mucosal immunity and airway memory t-cell responses are crucial players in protection against respiratory viruses, since these areas are the first to encounter the virus. therefore, along with selecting antigens for a vaccine, the route of vaccination and adjuvants are key players that cannot be neglected in vaccine design. because the spike protein and more specifically the s1 domain, is highly divergent among different covs, neutralizing antibodies only provide homotypic protection. thus far, the variability in the amino acid sequence of the spike protein observed among mers-cov strains is low [71] , and circulating mers-cov strains did not show any significant variation in the serological reactivity [25, 59] , implying that the development of a vaccine that is effective against one strain is likely to be protective against all circulating strains. another risk posed from the development of antibody escape variants is still present [72, 73] , although this is not likely to happen as mutations in two epitopes may be required, and mutants that develop may have reduced viral fitness [73, 74] . while the rbd is considered an ideal vaccine candidate for mers-cov, the spike s2 domain and n protein are more conserved, and thus adaptive immune response directed against these proteins can potentially lay the basis for a more broadly acting coronavirus vaccine. however, evidence for cross reactive immune responses against different covs is limited to a few studies. convalescent sars-cov patient sera weakly neutralized mers-cov [75] and sars-s reactive antisera showed low level neutralization of mers-cov [61] . extra-rbd s1 or s2 epitopes could be responsible for this effect, as some neutralizing epitopes have been identified in these regions of the s protein [58, 62] . these may not be as immunodominant as the rbd epitopes but could provide a rationale for the development of a cross protective cov epitope-focused vaccine. a recent study also demonstrated the potential role of adaptive response against n protein in protection against mers-cov infection as this vaccine candidate produced a protective t-cell response against mers-cov challenge which was also partially protective against sars-cov [27 ] . moreover, infection of mice with sars-cov reduced mers-cov titers 5 days p.i. upon challenge suggesting the development of a cross reactive t-cell response [26] . thus, mapping and focusing the immune response towards these critical neutralizing and t-cell epitopes, which could be subdominant, may provide a way to induce immune responses with a broader activity against different covs. immune focusing may also be beneficial for the generation of a robust virus-specific immune response. as during vaccine preparation, some epitopes which are normally hidden in the full length protein structure get exposed. some epitopes could be immunodominant and have a negative contribution on the overall neutralization capacity produced by the vaccine [76 ] . this also holds true for some non-neutralizing immunodominant epitopes, as s1-based vaccines induced slightly higher neutralization than whole s ectodomain-based ones [58, 53] . additionally, the rbd induced higher neutralizing antibodies compared to an s1 subunit vaccine [48] , and shorter regions of rbd induced even higher neutralization responses [66] , indicating that additional regions inducing non-neutralizing antibodies may contribute negatively to the overall neutralization response produced. additionally, antibody-dependent enhancement of the viral infection by non-neutralizing antibodies [77] [78] [79] , despite not being reported so far for mers-cov, needs also to be taken into consideration when developing a coronavirus vaccine. one approach to enhance the efficacy of subunit vaccines is to mask those negativelycontributing epitopes through glycosylation [76 ] . other approaches are immunefocusing and epitope-based vaccines, all aiming at narrowing the immune response to target only critical or beneficial epitopes to produce a stronger protective response. a prerequisite to reach that goal is to map epitopes targeted by the immune system and identify their biological role as being neutralizing, non-neutralizing, infection enhancing, containing a tcell epitope, and so on. this can be achieved by analyzing the activity and fine specificity of convalescent patient sera, infected animal polyclonal sera, monoclonal antibodies, animal and human pbmcs. subsequently the predicted epitopes can also provide a basis for potential vaccine candidates when produced as nanoparticles or vlps. further characterization of the immune responses induced by these vaccine candidates when evaluated in an animal model may be utilized to optimize the vaccines for efficacy (figure 1 ). this epitope-focused vaccine approach may allow for targeting less immunodominant b-and t-cell epitopes having broader protection, avoid eliciting immune responses against epitopes playing no role in protection or having a negative or harmful role. in addition to better targeting of protective immunodominant epitopes, a combination of those epitopes, b-and tcell epitopes targeting different viral proteins, could be used to produce a broader and stronger protective immune response for both strain-specific and universal cov vaccines. next to the choice of the mers-cov vaccine candidate, it is also important to take into account the target population that needs to be protected through vaccination. populations at risk of mers-cov infection include camel contacts, healthcare workers and patient contacts. the latter two groups could benefit from the rapid onset of immunity though passive immunization using mabs or convalescent sera, provided that it is given in time. another alternative strategy for short-term protection is the use of vaccines capable of rapidly inducing high titers of neutralizing antibodies. this will provide a short-term immunity beneficial to protect those highly-at-risk groups when a new case is identified, to prevent outbreaks. to prevent virus infection of primary cases, vaccination of the dromedary camels may also be considered. so far, among the available vaccine candidates, only two have been tested in dromedary camels, pvax1-s and mva-s. pvax1-s, a dna-based vaccine, induced neutralizing antibodies in two of three camels tested so far, but has not been tested for efficacy [59] . the other candidate mva-s, a viral-vector-based vaccine, induced systemic neutralizing antibodies and mucosal immunity which conferred protection against mers-cov challenge and reduced virus shedding in vaccinated camels [52 ] therefore, this vaccine candidate may provide a means to prevent zoonotic transmission of the virus to the human population. for camel contacts and healthcare workers in endemic areas, being at a continuous risk of mers-cov infection either from infected camels or patients, respectively, it would be beneficial to induce a longer-term (mucosal) protection. while these could be rewarding approaches to stop mers-cov outbreaks, it is still worthwhile to develop platforms and vaccines that aim to induce more broad protection against different related covs, that could potentially cause future outbreaks. learning from previous epidemics, the who issued a list of priority pathogens posing a risk to the human population and requiring urgent research and development (r&d) for intervention measures, among which mers-cov and highly pathogenic covs are of high priority. the lack of intervention measures along with the increase in geographical area and ongoing mersepitope-based vaccine design. following a virus infection, potential protective b-and t-cell epitopes are mapped. peptides or proteins containing promising epitopes are produced and formulated using a suitable platform, for example, nanoparticles and tested for immunogenicity and efficacy in animals. follwing several cycles of testing and optimization, a final vaccine suitable for human use may be produced. cov cases, raise worries for the future occurrence of larger epidemics as a result of virus adaptation in the human population and more efficient human-to human transmission. further development of mers-cov and other cov vaccines thus needs proactive collaborative efforts from researchers filling knowledge gaps, and market stakeholders providing funding for this costly process. the latter can be insufficient and/or unsustainable, therefore hindering development of even some promising candidates. in an initiative aiming at accelerating vaccine r&d process by providing sustained funding to be prepared for future epidemics, the world economic forum launched the coalition for epidemic preparedness innovations (cepi) [80] . cepi is an international non-profit association aiming at removing barriers facing vaccine development for epidemic infections and getting ready for future epidemics, including mers-cov. however, we still face a number of challenges despite the fact that various promising mers-cov vaccine candidates are currently available. there is a lack of animal models mimicking the disease in humans in which vaccine platforms can be tested prior to human use. we need to take into account the populations to target with vaccination, with camels and camel handlers being the most relevant ones. the lack of full understanding of the pathogenesis and immune responses to the virus in humans and camels, which is crucial for vaccine development, also needs further investments. in addition, the longevity of immune responses post-vaccination has not been evaluated for vaccine candidates, which is important for the vaccination scheme development and for the choice of the best candidates for further development. lastly, most of the vaccine candidates are developed against the highly variable spike protein and thus may not be able to provide protection against cov strains evolving in the future. a more targeted vaccination approach aiming at conserved epitopes should be considered for the development of a more broadly-acting cov vaccine. given the propensity of covs to jump the species barrier, current efforts to develop a mers-cov vaccine may also be of benefit to prepare for potential novel covs that may emerge in the future. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest the immunobiology of sars* isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study occupational exposure to dromedaries and risk for mers-cov infection presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study unraveling the drivers of mers-cov transmission risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel transmission of mers-coronavirus in household contacts clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia animal models of middle east respiratory syndrome coronavirus infection a comparative review of animal models of middle east respiratory syndrome coronavirus infection 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012. virology prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome airway memory cd4(+) t cells mediate protective immunity against emerging respiratory coronaviruses this study shows the efficacy of an anti-nucleocapsid vaccine for mers-cov, t-cell mediated protection and potential cross protection, and the role of route of administration in protection haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection persistence of antibodies against middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death mortality associated with influenza and respiratory syncytial virus in the united states grubeck-loebenstein b: vaccines for the elderly mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice exacerbated innate host response to sars-cov in aged non-human primates evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus t cell-mediated immune response to respiratory coronaviruses review on the role of t-cells in protection against respiratory coronaviruses age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus. hum. vaccines immunother middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara delivering middle 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syndrome coronavirus in nonhuman primates mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice the amino acids 736-761 of the mers-cov spike protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents the recombinant nterminal domain of spike proteins is a potential vaccine against middle east respiratory syndrome coronavirus (mers-cov) infection recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection intranasal vaccination with recombinant receptorbinding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness 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vaccine candidate this work was supported by the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. 115760), with the assistance and financial support of imi and the european commission, inkind contributions from efpia partners. key: cord-263016-28znb322 authors: omrani, a.s.; shalhoub, s. title: middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? date: 2015-08-22 journal: j hosp infect doi: 10.1016/j.jhin.2015.08.002 sha: doc_id: 263016 cord_uid: 28znb322 the middle east respiratory coronavirus (mers-cov) was first isolated from a patient who died with severe pneumonia in june 2012. as of 19 june 2015, a total of 1,338 mers-cov infections have been notified to the world health organization (who). clinical illness associated with mers-cov ranges from mild upper respiratory symptoms to rapidly progressive pneumonia and multi-organ failure. a significant proportion of patients present with non-respiratory symptoms such as headache, myalgia, vomiting and diarrhoea. a few potential therapeutic agents have been identified but none have been conclusively shown to be clinically effective. human to human transmission is well documented, but the epidemic potential of mers-cov remains limited at present. healthcare-associated clusters of mers-cov have been responsible for the majority of reported cases. the largest outbreaks have been driven by delayed diagnosis, overcrowding and poor infection control practices. however, chains of mers-cov transmission can be readily interrupted with implementation of appropriate control measures. as with any emerging infectious disease, guidelines for mers-cov case identification and surveillance evolved as new data became available. sound clinical judgment is required to identify unusual presentations and trigger appropriate control precautions. evidence from multiple sources implicates dromedary camels as natural hosts of mers-cov. camel to human transmission has been demonstrated, but the exact mechanism of infection remains uncertain. the ubiquitously available social media have facilitated communication and networking amongst healthcare professionals and eventually proved to be important channels for presenting the public with factual material, timely updates and relevant advice. the middle east respiratory syndrome coronavirus (mers-cov) was first identified in september 2012. as of 19 june 2015, a total of 1,338 mers-cov infections have been reported to the world health organization (who). 1 despite an accumulation of clinical experience and scientific knowledge, new mers-cov infections continue to be reported almost on daily basis. what lessons can we learn after three years of clinical experience and scientific research? lesson one: no substitute for continuous vigilance a 60-year-old man was admitted on june 13th, 2012, to a hospital in jeddah, saudi arabia, with severe pneumonia and multi-organ failure. the patient died after 11 days of hospitalization. 2 indirect immunofluorescence assays and real-time polymerase chain reaction (pcr) for widely occurring respiratory viruses failed to identify an infective aetiology. interestingly, cytopathic changes consistent with viral replication were noted in llc-mk2 and vero cell cultures of the patient's respiratory samples. slides of the infected cell cultures reacted strongly with the patient's serum but not with any of 2400 control sera stored in the same hospital. however, pancoronavirus pcr yielded positive results. the pcr fragments were sequenced at the erasmus medical centre in rotterdam, the netherlands, and phylogenetic analysis showed that the novel coronavirus belonged to lineage c of the genus betacoronavirus. 2, 3 on 20 september 2012, an email was posted on program for monitoring emerging diseases mail (promed-mail) announcing the discovery of a novel human coronavirus. 4 meanwhile, a critically ill 49-year-old qatari man was transferred by air ambulance on september 11th, 2012, to a hospital in england. 5 he had developed respiratory symptoms on september 3rd followed by multi-organ failure. his upper and lower respiratory tract samples were negative for influenza a/b, parainfluenza 1e4, rsv a/b, human metapneumovirus, enterovirus, rhinovirus, adenovirus, human bocavirus, and the human coronaviruses (nl63, 229e, oc43, hku1). on september 21st, 2012, one day after the above promed-mail posting, the patient's respiratory samples tested positive by pancoronavirus pcr. once sequenced, a 250 base-pair fragment from this isolate showed 99.5% homology with the erasmus medical centre's isolate. 5 the third patient was a 45-year-old man who presented to a hospital in riyadh, saudi arabia, on october 10th, 2012, with severe pneumonia and renal failure. mers-cov was detected in samples from the patient's upper and lower respiratory tract. 6 prior to all of this, an outbreak of respiratory illness was reported in april 2012 from an intensive care unit in a hospital in zarqa, jordan. 7 a retrospective epidemiological investigation in november 2012 identified 13 probable cases, two of whom had died. mers-cov was detected by reverse transcription (rt)epcr in stored samples from the two deceased patients. 8 seven more were subsequently confirmed by serological testing. 9 a pattern began to emerge, characterized by severe pneumonia, multi-organ failure, and an epidemiological link to a country in the middle east. in may 2013, the virus, which had been initially known as human coronaviruseerasmus medical centre (hcov-emc), was named the middle east respiratory syndrome coronavirus (mers-cov). 10 notably, phylogenetic analysis of the first five available mers-cov sequences suggested a common ancestor dating back to mid-2011. 11 furthermore, anti-mers-cov antibodies were detected in 15 out of 10,009 serum samples [0.15%; 95% confidence interval (ci): 0.09e0.24%] obtained between december 2012 and december 2013 from 13 provinces in saudi arabia. 12 the authors extrapolated that just fewer than 45,000 individuals (44, 951; 95% ci: 26, 971e71, 922) in saudi arabia could be seropositive for mers-cov. 12 it is therefore reasonable to assume that human mers-cov infections had taken place in the region for some considerable time before it was identified. it is possible that the identification of the virus might have been delayed even more, had it not been for the meticulous investigation by a single virologist, dr a.m. zaki, of the first reported case of mers-cov. 2 the first lesson one has to learn from mers-cov and its discovery is that continuous vigilance and perseverance with diagnostic investigation of undiagnosed infectious diseases are essential to identify emerging pathogens. lesson two: yet again, prevention is better than cure clinically, mers-cov infection may range from an asymptomatic or mild upper respiratory illness to a rapidly progressive and fatal disease. 13e15 the majority of hospitalized patients with mers-cov infection present with fever and respiratory symptoms including cough and shortness of breath with clinical and radiological evidence of pneumonia. 16,17 fatigue, myalgia, headache, and gastrointestinal symptoms such as vomiting and diarrhoea are also frequent. 16, 17 respiratory and renal failure are frequent complications of severe mers-cov infection, in addition to acute liver injury, cardiac dysrhythmias, and coagulopathy. 16e20 overall mortality is around 35.6%, but exceeds 70% in critically ill patients and in those with significant comorbidities. 1,18e22 for reasons yet to be understood, mers-cov infection is rare in children. 23, 24 in-vitro studies have identified numerous agents with anti-mers-cov activity including interferon, ribavirin, mycophenolate, cyclosporine and lopinavir. 25 the combination of interferon and ribavirin showed promising results in experimentally infected macaques. 26 however, in retrospective clinical studies the combination was not associated with significantly improved overall survival. 21,22 treatment of patients with mers-cov infections remains largely dependent on supportive measures. 27 diagnosis is confirmed by detection of mers-cov rna in respiratory samples by real-time pcr targeting the upe and orf 1b genes. 28 samples obtained from the lower respiratory tract have higher viral loads and better diagnostic yield than those obtained from the throat or nasopharynx. 11,29,30 moreover, viral shedding is considerably prolonged in symptomatic and severely ill mers-cov patients compared with asymptomatic infected contacts. 31 interestingly, detection of mers-cov in blood has been associated with worse clinical outcome. 22,32 mers-cov may also be detected in stool for up 16 days and in urine for up to 13 days from disease onset. 11 under certain conditions, mers-cov can survive on plastic and steel surfaces for up to 30 h. 33 in the absence of appropriate precautions, the environment surrounding a symptomatic mers-cov patient can therefore become extensively contaminated with viable, potentially infectious virus. human-to-human transmission of mers-cov has been well documented in family clusters, community settings and more often in healthcare settings. 13e15,17,20,34 common denominators in the largest hospital outbreaks have been overcrowding, especially in emergency departments, and poor adherence to infection control standards. 15,17,35,36 however, mers-cov continues to have relatively limited infectiousness. for example, screening identified secondary mers-cov infections in only 4% of 280 close family contacts and 2% of 5065 healthcare contacts. 37,38 moreover, no secondary cases were identified following extensive epidemiological investigations of imported cases in the uk, germany, france, greece, the netherlands, and the usa. 39e46 it has been phylogenetically demonstrated that mers-cov transmission chains have not extended beyond two to three months and that the virus has remained genetically stable over the past three years. 47, 48 given an effective reproduction number (r 0 ) of less than one, human-to-human mers-cov could be readily interrupted with effective preventive interventions. 49, 50 indeed, even the most explosive hospital outbreaks of mers-cov infection, such those that occurred in jeddah and riyadh in april to may 2014, were brought under control through a strategy based on early case detection and implementation of appropriate infection prevention and control measures; namely contact and droplet precautions for general care in addition to airborne precautions for aerosolgenerating procedures such as intubation and respiratory tract suctioning. 51e53 the poor prognosis associated with mers-cov, especially in patients with multiple comorbidities, and the lack of effective anti-viral therapy make appropriate infection prevention and control all-important. just as is true for most infectious diseases, mers-cov reminds us again that prevention is better than cure. the initial case definitions for mers-cov case finding and reporting focused on patients who are hospitalized, had evidence of acute pulmonary disease with an epidemiological link to confirmed cases or to countries in the middle east. 54, 55 as more clinical experience and epidemiological data became available, updated definitions removed the requirement for hospitalization. 56 the reporting of several community and hospital clusters during the first half of the year 2013, often without identifiable human or animal sources, led to speculation that individuals with no or only mild respiratory symptoms might have a role in mers-cov transmissions. 13, 14, 20 this was reflected in the who revised interim definition published in july 2013 where patients with acute febrile illness of any severity were included; in addition to a recommendation to proactively test asymptomatic close contacts of confirmed mers-cov infections. 57 memish et al. later showed that mers-cov was detectable for up to 12 days in 30% of 12 asymptomatic contacts. 31 in another report, an asymptomatic healthcare worker had detectable mers-cov for more than five weeks. 58 although mers-cov transmission from an asymptomatic individual remains a strong probability, this has never been documented. 37, 38 in the meantime, clinicians were becoming increasingly aware that mers-cov infections were being diagnosed in patients whose clinical presentations did not conform to those definitions, including the absence of fever, lack of respiratory involvement and the predominance of gastrointestinal or nonspecific generalized symptoms. 17, 22, 59 in the aftermath of the surge of mers-cov infection in jeddah and riyadh in april and may 2014, the ministry of health in saudi arabia revised its case definition and surveillance guidance to recommend mers-cov testing in any of four patient categories: e patients with clinical or radiological evidence of community-acquired pneumonia; e patients with clinical or radiological evidence of healthcare-associated pneumonia; e patients with acute febrile illness and myalgia, headache, diarrhoea, nausea, or vomiting, and unexplained leucopenia or thrombocytopenia; e contacts of individuals with confirmed or probable mers-cov infection who develop upper or lower respiratory symptoms within two weeks of exposure. 60 as better understanding of the epidemiology of mers-cov developed, it became obvious that a considerable proportion of cases were probably missed. 12,50 during the steep learning curve of an emerging infectious disease, regularly updated guidelines are important. such guidelines are inevitably based on incomplete evidence and hence may not be comprehensive or applicable in all situations. clinical acumen and heightened medical awareness are essential for early detection of unusual mers-cov cases and to prevent delays in diagnosis and to mitigate additional exposures. 61 a zoonotic origin was suspected soon after the identification of mers-cov. 6 bats are known natural hosts for several coronaviruses and hence were the initial target for investigation. 62, 63 more than 1000 faecal samples were collected from wild bats in the area around where the first mers-cov patient lived. a 190-nucleotide fragment of mers-cov rna was detected in one faecal pellet from an egyptian tomb bat. the sequenced amplification product was genetically identical to the mers-cov sequence obtained from the index human case. 64 more recently, a closely related coronavirus was isolated from bats in south africa, suggesting that mers-cov ancestors might exist in old world bats. 65, 66 to date, no further evidence is available to confirm the role of bats as natural hosts or reservoirs for mers-cov. on the other hand, the evidence implicating dromedary camels in mers-cov epidemiology is more consistent. a role for dromedary camels is supported by the following observations: à neutralizing mers-cov antibodies are highly prevalent in dromedary camels from across the arabian peninsula, north africa, and eastern africa. 67e73 mers-cov antibodies were detected in stored camel sera dating as far back as the early 1990s. 73e75 the prevalence of mers-cov seropositivity is significantly higher in camels aged more than two years than in juvenile camels. 68,74,76 à several groups have reported the detection of mers-cov by rtepcr in nasal and faecal samples from dromedary camels in the arabian peninsula. 72,74,76e79 one study reported mers-cov positivity in more than 60% of lung tissue samples obtained from dromedary camel carcasses. 79 rtepcr was positive in camels that had prior evidence of mers-cov seropositivity, indicating that animal reinfection is possible. 76 interestingly, the prevalence of mers-cov rna is significantly higher in juvenile than in adult camels. 74, 76, 79 furthermore, all mers-cov strains obtained from dromedary camels are phylogenetically clustered within human isolates, supporting possible animalehuman intertransmission. 72 it is important to note, however, that mers-cov seroprevalence studies in individuals with close contact with camels have yielded inconsistent results. a national serosurvey in saudi arabia found prevalence of mers-cov antibodies that was 15 times higher in camel shepherds (p ¼ 0.0004) and 23 times higher in slaughterhouse workers (p < 0.0001), compared with the general population. 12 similarly, mers-cov serology was positive in individuals who had occupational exposure to dromedary camels in qatar but not in those without such exposure. 86 on the other hand, mers-cov antibodies were not detected in sera obtained from individuals who had close contact with camels that had documented mers-cov infection two to three months earlier. 87 likewise, screened slaughterhouse workers and other animal workers in western and southern saudi arabia were all seronegative for mers-cov antibodies. 88, 89 collectively, the available data strongly suggest that mers-cov is highly prevalent in dromedary camels in the arabian peninsula and that transmission of infection from camels to humans, although inefficient, does occur. however, the exact mechanism and route of infection it is still unclear. infections. 30, 94, 95, 97 one pertinent cause for concern has been the potential global spread of mers-cov during the annual hajj pilgrimage when millions of muslims from around the world gather in mecca, saudi arabia. 98e102 though those concerns are well founded, several surveillance studies over the past three years have not identified any mers-cov infections among hajj pilgrims while they are in saudi arabia or after their return to their home countries. 103e108 the situation was entirely different in the recent outbreak in south korea where a single imported case resulted in a total of 186 laboratory-confirmed cases of mers-cov infection, including 36 deaths. 109 the index patient was a 68-year-old man who developed respiratory symptoms seven days after returning to seoul from a two-week visit to bahrain, saudi arabia, united arab emirates, and qatar. 35 he sought medical care in several hospitals before he was diagnosed with mers-cov infection. 35,110 a combination of late recognition, overcrowding in emergency departments and hospital wards, multiple incidents of patient movement between different healthcare facilities, and delayed implementation of adequate infection control precautions culminated in the largest single outbreak of mers-cov infection. 35,36,110e112 the outbreak involved patients, visitors, care-givers and healthcare workers, and spanned across six different hospitals in three south korean cities. 110, 113 notably, phylogenetic analysis of mers-cov strains from south korea revealed no significant biological changes compared to previously sequenced viruses. 114 the outbreak in south korea was eventually controlled through a series of measures including aggressive contact identification, screening and strict isolation, and rigorous infection control precautions. 111, 115, 116 within a few weeks, south korea went from a country with no reported mers-cov cases to one that has the second largest number in the world. 116, 117 with air travel becoming readily accessible and affordable, the south korean experience demonstrates vividly that in the context of an infectious respiratory illness, there is simply no room for complacency. adequate assessment of patients presenting with febrile illness must include their recent travel history to enable early application of proper control measures and to expedite laboratory confirmation and appropriate clinical management. the past decade has witnessed an exponential rise in internet-based social media sites such as facebook, twitter, and youtube. 118 healthcare professionals are increasingly using social media applications to follow medical developments and emerging scientific literature and to share their own research findings, observations, and opinion. 119 the general public often uses these tools as news outlets to seek and share medical and scientific information. 120 however, in the context of mers-cov, social media have been a double-edged sword. for example, social media were at some point rife with inaccurate information that included rumours of hospitals closed due to mers-cov outbreaks and certain social events being nodes for mers-cov transmission. the authors are aware of examples of information and photos shared on social media resulting in patients losing their right to privacy and confidentiality. patients often cancelled their clinic appointments or scheduled surgical procedures for fear of acquiring mers-cov while in hospital. some avoided attending emergency departments despite having acute problems that required medical attention. some individuals posted videos and messages challenging the suggestion that camels may be a source of mers-cov infection. scepticism and mistrust in governmental agencies and the medical community were sometimes promoted and propagated. on the other hand, various government agencies, scientific organizations and healthcare professionals used social media to enhance networking and facilitate communication of epidemiological, medical and scientific developments; in addition to presenting the public with factual material, timely updates, and relevant advice. 121 the saudi ministry of health, for example, posts daily updates on its website and through social media outlining details of current mers-cov cases. the korean ministry of health and welfare did the same during their mers-cov outbreak. such steps are important to gain the public's trust and to remove barriers to appropriate sources of information. taking on board the surging role of social media and using them effectively to disseminate appropriate information turns them into invaluable tools for controlling an emerging infectious disease such as mers-cov. mers-cov is an emerging infectious disease of probable animal origin. sustained human-to-human infection has not occurred and its potential for causing widespread epidemic remains limited. vigilance, early recognition, and institution of appropriate protective measures are the most effective control measures. none declared. middle east respiratory syndrome coronavirus (mers-cov); summary of current situation, literature update and risk assessment 7 isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans novel coronavirus e saudi arabia: human isolate. archive number severe respiratory illness caused by a novel coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) investigations and implications for the prevention of human-to-human transmission a case of imported middle east respiratory syndrome coronavirus infection and public health response middle east respiratory syndrome coronavirus 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patients with middle east respiratory syndrome coronavirus (mers-cov) infection world health organization. case definition for case finding severe respiratory disease associated with novel coronavirus. interim case definition as of 25 revised interim case definition e novel coronavirus. interim case definition as of 16 revised interim case definition for reporting to who e middle east respiratory syndrome coronavirus (mers-cov) revised interim case definition for reporting to who e middle east respiratory syndrome coronavirus (mers-cov) a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker successful recovery of mers-cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report case definition and management of patients with mers coronavirus in saudi arabia investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov); interim guidance updated 3 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camels antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity mers coronavirus in dromedary camel herd, saudi arabia mers coronaviruses in dromedary camels isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia acute middle east respiratory syndrome coronavirus infection in livestock dromedaries tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus occupational exposure to dromedaries and risk for mers-cov infection lack of middle east respiratory syndrome coronavirus transmission from infected camels investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012 middle east respiratory syndrome coronavirus (mers-cov) e thailand. disease outbreak news 20 middle east respiratory syndrome coronavirus (mers-cov) e austria clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus (mers-cov) e the philippines investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in family cluster of middle east respiratory syndrome coronavirus infections laboratoryconfirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response health protection agency uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj middle east respiratory syndrome (mers) coronavirus. what travel health advice should be given to hajj pilgrims preventive measures against mers-cov for hajj pilgrims the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj from the hajj: it's the flu, idiot high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage saudi arabia, south korea preliminary epidemiological assessment of mers-cov outbreak in south korea lessons to learn from mers-cov outbreak in south korea spread of mers to south korea and china list of hospitals with known mers exposure preliminary data from sequencing of viruses in the republic of korea and the people's republic of china. mers-cov situation assessment 9 world health organization. managing contacts in the mers-cov outbreak in the republic of korea geneva: who. available at middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china e as of 19 korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea dangers and opportunities for social media in medicine social media and health care professionals: benefits, risks, and best practices social media and clinical care: ethical, professional, and social implications middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd key: cord-255488-nvgz53su authors: li, kun; mccray, paul b. title: development of a mouse-adapted mers coronavirus date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_13 sha: doc_id: 255488 cord_uid: nvgz53su first identified in 2012, middle east respiratory syndrome coronavirus (mers-cov) is a novel virus that can cause acute respiratory distress syndrome (ards), multiorgan failure, and death, with a case fatality rate of ~35%. an animal model that supports mers-cov infection and causes severe lung disease is useful to study pathogenesis and evaluate therapies and vaccines. the murine dipeptidyl peptidase 4 (dpp4) protein is not a functional receptor for mers-cov; thus, mice are resistant to mers-cov infection. we generated human dpp4 knock-in (hdpp4 ki) mice by replacing exons 10–12 at the mouse dpp4 locus with exons 10–12 from the human dpp4 gene. the resultant human dpp4 ki mice are permissive to mers-cov (hcov-emc/2012 strain) infection but develop no disease. to generate a mouse model with associated morbidity and mortality from respiratory disease, we serially passaged hcov-emc/2012 strain in the lungs of young hdpp4 ki mice. after 30 in vivo passages, an adapted virus clone was isolated and designated mers(ma)6.1.2. this virus clone produced significantly higher titers than the parental clone in the lungs of hdpp4 ki mice and caused diffuse lung injury and a fatal respiratory infection. in this chapter, we will describe in detail the procedures used to mouse adapt mers-cov by serial passage of the virus in lungs. we also describe the methods used to isolate virus clones and characterize virus infection. middle east respiratory syndrome (mers) is a fatal respiratory illness that first appeared on the saudi arabian peninsula in mid-2012. it is caused by a novel betacoronavirus, mers coronavirus (mers-cov) [1] . shortly after the identification of the virus, its receptor dipeptidyl peptidase 4 (dpp4) was discovered [2] . as of september 2019, the world health organization (who) has reported 2468 laboratory-confirmed cases of mers in 27 countries, including 851 associated deaths (fatality rate: 35%). mers-cov does not currently have pandemic potential [3] [4] [5] . however, mers-cov is still epidemic in the middle east and remains a cause for significant concern due to the potential spread by global travel as demonstrated by the outbreak in south korea in 2015 [6] [7] [8] . to date, there are only two published reports on autopsy findings from subjects who died from mers [9, 10] and our understanding of mers-cov pathogenesis in humans is still limited. animal models are useful for the study of viral diseases and play an important role in the investigation of pathogenesis and evaluation of antiviral therapies and vaccines. an ideal animal model should be permissive to the viral infection and develop disease and pathology with similarities to that observed in humans. mers-cov infection has been evaluated in two nonhuman primate (nhp) models, the rhesus macaque and common marmoset [11] [12] [13] [14] [15] . both species are susceptible to mers-cov infection. however, mers-cov caused only a transient lower respiratory tract infection without mortality in rhesus macaques [11] [12] [13] . in common marmosets, the consequences of mers-cov infection are controversial. falzarano et al. reported the common marmoset reproduced several features of mers-cov infection in humans including progressive severe pneumonia [15] , while another group observed only mild to moderate nonlethal respiratory disease following mers-cov infection [14] . thus, the common marmoset is potentially a model to study pathogenesis and evaluate antiviral therapies and vaccines. however, nhps are expensive, their availability limited, and their use may raise ethical concerns. in contrast, small animal models provide advantages over nhps, including reduced cost, availability in large numbers, ease of handling, and species-specific reagents, especially for the studies of highly pathogenic viruses like mers-cov in the biosafety level 3 (bsl-3) laboratory. unfortunately, common small laboratory animals like mice [16, 17] , ferrets [18] , guinea pigs [19] , and hamsters [20] are not susceptible to mers-cov infection because their homologous dpp4 cannot be bound and utilized by mers-cov as a host receptor for entry [21, 22] . haagmans et al. detected mers-cov rna in the respiratory tract of new zealand white rabbits following inoculation but found no clinical signs of disease [23] . several strategies have been used to overcome this receptor incompatibility and develop mouse models of mers-cov infection. in 2014, we developed the first mouse model of mers-cov infection [24] . we delivered a recombinant adenovirus 5 encoding human dpp4 (hdpp4) to the lungs of mice. transient expression of hdpp4 by adenovirus transduction made the mice temporarily permissive for mers-cov infection, but animals developed only mild lung disease. generation of transgenic mice expressing a virus receptor is a common strategy to make mice permissive to infection. several groups in addition to ours developed mice with transgenic expression of hdpp4 using different promoters [25] [26] [27] [28] . the disease severity following mers-cov infection in transgenic mice correlated with the cellular distribution and expression level of hdpp4. in transgenic mice expressing hdpp4 driven by the cytokeratin 18 promoter [26] or a ubiquitous promoter [25, 27, 28] , mers-cov replicates and causes respiratory disease and mortality. however, the lethality was found to be secondary to overwhelming central nervous system (cns) disease or multiorgan damage. this was also observed in mice transgenic for human ace2 driven by the cytokeratin 18 promoter and infected with sars-cov [29] . in transgenic mice expressing hdpp4 under the human surfactant protein c (spc) promoter, which restricts expression to bronchiolar and alveolar epithelia, mers-cov infection caused only mild disease [26] . thus, these transgenic mice do not reproduce a severe lung disease phenotype that resembles mers. alternative strategies for the creation of mouse models of mers-cov infection are generation of dpp4 humanized mice and adaptation of the virus to the animals. pascal et al. reported a model in which all of the mouse dpp4 exons had been humanized and also generated humanized monoclonal antibodies against the mers-cov s protein using a novel strategy [30] . mers-cov infection in this model caused pulmonary edema, vascular cuffing, and alveolar septal thickening with an associated~20% weight loss, necessitating euthanasia [31] . another mers mouse model was engineered by changing two amino acids in the mouse dpp4 locus using crispr-cas9 technology [32] . this model supported mers-cov replication without severe disease. similarly, our human dpp4 knock-in mouse model supported mers-cov replication but did not lead to a severe lung disease phenotype [33] . two mouse-adapted (ma) strains of mers-cov were subsequently developed independently by serial passage of the hcov-emc/2012 strain [1] in the lungs of the two humanized mouse models [32, 33] . the resultant mers-15 and mers ma 6.1.2 mouse-adapted mers-cov strains replicated to high titers in the lungs of the crispr-cas9 genetically engineered mouse model and the hdpp4 knock-in mouse model, respectively. the respiratory disease that developed in both mouse models and the associated mortality shared similarities with severe cases of mers [32, 33] . mouse adaptation was also successfully used to generate several sars-cov strains capable of modeling severe sars-cov lung disease in mice [34] [35] [36] . thus, the adaption of the virus to enhance virulence in the mouse is a very useful approach to generate mouse models for coronavirus-associated lung disease. these materials can be altered to fit the requirements for other viruses of interest. 1. hcov-emc/2012 strain. 2. hdpp4 knock-in mouse (c57bl/6 strain with mouse dpp4 exons 10-12 replaced with the human codons). all procedures are performed under bsl-3 laboratory conditions and must follow the standard operating protocol of a bsl-3 facility and regulatory agencies. all manipulations of infectious specimens, samples, and mice must be performed within a biosafety cabinet or within a contained device such as a centrifuge. all tissue culture media and waste must be bleached and autoclaved prior to disposal. all instruments must be disinfected with virex plus or 10% bleach. 5. insert surgical scissors under the sternum and cut the diaphragm following the costal arch. remove the rib cage using scissors and forceps, exposing the lungs and heart. 6. fill a 10 ml syringe with cold sterile dpbs using a 25 g â 5/8 inch needle. insert the needle into the apex of the left ventricle and make a small incision in the right atrium. slowly perfuse !5 ml cold sterile dpbs into the left ventricle. next, insert the needle into the apex of the right ventricle and perfuse !5 ml cold sterile dpbs (see note 10). 7. remove the lungs and heart from the thoracic cavity. remove the liver, kidney, spleen, and small intestine. place organs in a small polystyrene weighing dish. remove remaining connective tissue. 8. to harvest the brain, turn the mouse over and wet the fur of the head with 70% ethanol. grasp the ears with forceps and cut off the skin and fur to expose the skull. remove remaining skin at the base of the neck to further expose the skull. immobilize the mouse and make an incision along the sagittal suture of the skull using a single edge razor blade (see note 11) . wedge one prong of the curved serrated forceps into the now opened sagittal suture. slowly pry up the skull, grasp the piece of skull with forceps and peel outward to remove. repeat on the other side. use curved forceps to lift the brain from the skull. place brain tissue in the small polystyrene weighing dish. 9 . carefully place each organ into a 50 ml disposable tissue grinder filled with 2 ml sterile cold dpbs for homogenization or into 2 ml of trizol for rna extraction (see note 12). 10. grind the lung tissue and transfer the homogenates or rna samples into 2.0 ml sterile screw-top tubes with o-ring cap (see note 13) . store samples at à80 c. thaw and spin down the cell debris in lung homogenates before use. the virulence of the virus should be evaluated in groups of mice by weight loss and survival after every 5-10 in vivo passages. after the virulence of the virus has been significantly enhanced, single plaques of the adapted virus should be purified and evaluated. 1. plate vero81 cells in d10 media in 6-well plates one day before infection. 2. rapidly thaw lung homogenates from the selected passage. mix the lung homogenates from two mice in a 2.0 ml sterile screwtop tube with o-ring cap on ice. 3. serially dilute the mixed lung homogenates tenfold in 2.0 ml sterile screw-top tubes with o-ring caps, using ice-cold serumfree dmem (see note 15) . keep the dilutions on ice. 4. remove the medium from each well and add diluted samples (in a volume of 400 μl) to each well. 5. place the plates in the 37 c incubator for 1 h and rotate gently every 15 min. 6. melt 2% low melting point agarose and maintain in a 65 c water bath. 7. mix overlay media and 2% low melting point agarose at a volume ratio of 1:1. rotate the tube several times to fully mix. overlay cells with 1.5 ml of mixed media using 10 ml stripettes. 8. let the plates sit in the hood for~5 min at rt or until the agarose overlay turns solid. add 0.5 ml d2 medium on the top of solidified agarose. 9 . place the plates in the 37 c incubator. 10 . after 3 days, plaques should be visible. remove the liquid on the top of the agarose. circle the visible plaques on the underside of the plates using a permanent marker (see note 16). 11. vertically penetrate the agarose and pipette the circled plaque several times with a 1 ml graduated transfer pipette. the agarose above the plaque will be pulled into the pipette. 12. transfer the agarose above the plaque into a 15 ml conical tube filled with 500 μl dmem by pipetting up and down several times. 13. transfer the 500 μl dmem containing the agarose into a 2.0 ml sterile screw-top tube with o-ring cap. 14. repeat the procedure and pick six single plaques. store the tubes at à80 c. 8. animal euthanasia should follow the institution's animal care and use guidelines. there may be differences in institutional requirements regarding when euthanasia is required based on weight loss. 9 . use the lid of an insulated foam shipping box. cut absorbent bench underpad (42 â 58 cm) into small pieces that fit the lid. 10 . carefully keep the tip of the needle in the lumen of the ventricle. be sure to puncture the right atrium as this will help to drain blood during the perfusion. the lung will turn white after perfusion. 11. make sure that the incision does not exceed the thickness of the skull; avoid cutting into the brain tissue. 12. the individual organs can be divided into pieces and transferred to grinders with pbs or trizol separately. 13. lung homogenates are immediately transferred to tubes on ice. rna samples are transferred to a tube and incubated for at least 15 min at room temperature per our bsl-3 specific standard operating procedures. 14. at 2 days post infection with 10 5 pfu/mouse, hcov-emc/ 2012 strain replicates in the lung of the hdpp4 ki mice to a titer of around 4 â 10 6 pfu/ml, which equals 2 â 10 5 pfu in 50 μl. we chose the 10 5 pfu/mouse inoculum to begin because we wanted to use a similar dose range during in vivo serial passage while skipping the titration step. 15. titrate the homogenates of the passage of interest and select a virus dilution that produces 10 plaques in a well. this allows identification of individual clear single plaques. 16 . only circle the unambiguous clear single plaques. 17. compared to vero 81 cells, we found that the mers-cov rna genome is more stable when propagated in huh7 cells (less likely to introduce genomic deletions, insertions, or point mutations). isolation of a novel coronavirus from a man with pneumonia in saudi arabia dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk person-toperson spread of the mers coronavirus-an evolving picture the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission spread of mers to south korea and china infectious diseases epidemic threats and mass gatherings: refocusing global attention on the continuing spread of the middle east respiratory syndrome coronavirus (mers-cov) comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease infection with mers-cov causes lethal pneumonia in the common marmoset mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus domestic pig unlikely reservoir for mers-cov the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection asymptomatic middle east respiratory syndrome coronavirus infection in rabbits rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a mouse model for mers coronavirus-induced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease we thank chris wohlford-lenane and jennifer bartlett for careful review of the manuscript. this work is supported by the national institutes of health p01 ai060699. we also acknowledge the support of the cell morphology core and pathology core, partially supported by the center for gene therapy for cystic fibrosis (national institutes of health p30 dk-54759) and the cystic fibrosis foundation. p.b.m. is supported by the roy j. carver charitable trust. key: cord-284374-sqxlnk9e authors: park, jiyeon; yoo, seung yeon; ko, jae-hoon; lee, sangmin m.; chung, yoon joo; lee, jong-hwan; peck, kyong ran; min, jeong jin title: infection prevention measures for surgical procedures during a middle east respiratory syndrome outbreak in a tertiary care hospital in south korea date: 2020-01-15 journal: sci rep doi: 10.1038/s41598-019-57216-x sha: doc_id: 284374 cord_uid: sqxlnk9e in 2015, we experienced the largest in-hospital middle east respiratory syndrome (mers) outbreak outside the arabian peninsula. we share the infection prevention measures for surgical procedures during the unexpected outbreak at our hospital. we reviewed all forms of related documents and collected information through interviews with healthcare workers of our hospital. after the onset of outbreak, a multidisciplinary team devised institutional mers-control guidelines. two standard operating rooms were converted to temporary negative-pressure rooms by physically decreasing the inflow air volume (−4.7 pa in the main room and −1.2 pa in the anteroom). healthcare workers were equipped with standard or enhanced personal protective equipment according to the mers-related patient’s profile and symptoms. six mers-related patients underwent emergency surgery, including four mers-exposed and two mers-confirmed patients. negative conversion of mers-cov polymerase chain reaction tests was noticed for mers-confirmed patients before surgery. mers-exposed patients were also tested twice preoperatively, all of which were negative. all operative procedures in mers-related patients were performed without specific adverse events or perioperative mers transmission. our experience with setting up a temporary negative-pressure operation room and our conservative approach for managing mers-related patients can be referred in cases of future unexpected mers outbreaks in non-endemic countries. high-volume healthcare facility, which is how the previous south korea outbreak occurred 11 . moreover, there may be very few hospitals that are prepared to provide perioperative care for mers patients. therefore, herein, we share our experience of providing infection prevention and control measures for surgeries for mers-related patients in our hospital. during the mers outbreak in our hospital, six mers-related patients underwent surgery including three possibly exposed patients, one directly exposed patient, and two mers-confirmed patients who recovered from the disease. all patients were negative during two preoperative mers screenings using real-time reverse transcription polymerase chain reaction (rrt-pcr) 14 . figure 1 shows the total number of surgeries performed during the outbreak period at our hospital and the distribution of mers-related patients undergoing surgery. temporary set-up of a negative-pressure operating room. the operating rooms in our hospital were generally positive-pressure environments, and we had no permanent negative-pressure operating rooms. because a negative-pressure operating room is the optimal environment to prevent airborne virus spreading to adjacent areas 13 , two of our 25 operating rooms in the main operating suite of the hospital were temporarily converted into negative-pressure operating rooms to perform surgical procedures on mers-related patients. operating rooms no. 16 and 17 were selected because they were connected to each other, but each room had separate atmospheric air inlets and exhaust systems. they also had separate air-conditioning and humidification systems. of the two connected rooms, one was used as the main operating room and the other was used as the anteroom where healthcare workers (hcws) applied and removed the personal protective equipment (ppe). in each room, fresh air was supplied from an inlet duct and discharged outside through the exhaust duct (fig. 2) . because a constant exhausting air volume was maintained through the outlet duct, negative pressure in the operating room was achieved by decreasing the inflow air volume that entered through the inlet duct. first, the blades of the air volume control damper in the inlet duct were closed as much as possible (fig. 2) . however, because the damper was not intended to be air-tight, the inflow volume to the operating room did not decrease sufficiently. second, as an additional measure to decrease the inflow volume, we opened the access hole in the inlet duct, which was originally used for duct inspection purposes (fig. 2) . finally, a smoke test was carried out to ensure negative pressure. the room pressure was maintained at −4.7 pa in the main operating room and at −1.2 pa in the anteroom (fig. 3) ; −4.7 pa is below the negative pressure room standard of −2.5 pa 15 . www.nature.com/scientificreports www.nature.com/scientificreports/ airflow in both rooms reached 14-18 air exchanges per hour, according to airflow velocity measurements with an anemometer (ebt731 balometer; tsi alnor ® , minnesota, united states). in this environment, removing airborne contaminants requires 18 minutes for 99% efficiency and 28 minutes for 99.9% efficiency 16 . therefore, 30 minutes of room ventilation was required after aerosol forming high-risk procedures, such as endotracheal intubation or extubation 12, 17 . the cleanliness level of each room was also measured using a particle counter (tsi 9310; tsi, united states): main operating room = 2,604 and the anteroom = 2,540, which were much lower than the institutional target level of <10,000 for general surgery (fig. 3) . cleanliness level was defined as the number of particles smaller than 0.5 µm in 0.3048 m 3 . equipment preparation and disinfection. all built-in instruments such as computers, telephones, and ventilators were covered with plastic paper. sufficient amounts of drugs, fluids, and other equipment were prepared in the operating room before surgery, and other unnecessary equipment was moved out. additionally, we used disposable equipment, when possible. high efficiency particulate air (hepa) filters were installed in the breathing circuits, both on the inspiratory and expiratory limbs of the ventilators and at the patient's site that connected to endotracheal tube. after operations with mers-exposed patients, 30 minutes of room ventilation was followed by surface disinfection with diluted chlorine bleach (500 ppm) 18, 19 . cleaners wore standard ppe while disinfecting surfaces. for mers-confirmed patients, surface disinfection was performed twice. institutional guidelines for perioperative management of mers-related patients. during the mers outbreak, we set the following principles for perioperative management of mers-related patients: all elective surgeries for mers-confirmed patients were postponed to reduce the risk of potential in-hospital transmission. for mers-exposed patients, surgical procedures were delayed until after the potential incubation period of 14 days 20 . when a mers-related patient required an urgent or emergency operation, mers-cov pcr tests were performed twice with distinct specimens preoperatively, to account for asymptomatic mers patients or delayed positive conversion in symptomatic mers-exposed patients. for patients with ambiguous pcr results or without a pcr test, operations were performed according to the management guidelines for mers-confirmed patients. all the surgical procedures for mers-related patients were performed in the last order of the day as possible. perioperative protection level for hcws. when an operation for a mers-related patient was scheduled, the division of infectious diseases and infection control department confirmed the protection level of the hcws, according to institutional guidelines (table 1 ). in principle, standard ppe was applied to hcws who cared for asymptomatic mers-exposed patients. standard ppe includes surgical gloves, surgical gowns, eye shields, and n95 respirators. while managing mers-confirmed or mers-exposed patients with mers-associated symptoms including fever, myalgia, respiratory symptoms, or diarrhea, hcws implemented enhanced ppe, which included coverall clothes with head cover, shoe covers, goggles, two pairs of surgical gloves, and powered air purifying respirator (papr) or n95 respirators. although we performed preoperative mers-cov pcr screening, enhanced ppe was still recommended when managing symptomatic mers-exposed patients regardless of their pcr results. anesthesiologists were recommended to apply enhanced ppe (including papr from the middle of the outbreak) when managing all mers-related patients because they were most directly exposed to the aerosol-producing high-risk procedures, such as endotracheal intubation and extubation. only minimal numbers of hcws were present in the operating room. institutional education regarding the precise use of ppe was provided to the all associated hcws and they were assisted by skilled nurses in the operating room during the ppe donning and doffing processes. patient transfer for operation. mers-related patients were transferred directly to the negative-pressure main operating room through an exclusive path and elevator by a physician wearing proper ppe. the walls and the floor of the passageways and the elevator were covered with plastic paper. mers-related patients wore a www.nature.com/scientificreports www.nature.com/scientificreports/ surgical mask during transfer. because only anesthesiologists wore enhanced ppe when in proximity to asymptomatic mers-exposed patients, 30 minutes of room ventilation was performed after anesthetic induction, including endotracheal intubation. the surgical team then entered the main operating room through the anteroom. in the cases of symptomatic mers-exposed patients or mers-confirmed cases, all hcws wore enhanced ppe and the 30-minute ventilation time was not required. after completion of operation procedures, patients who were moved to the general ward recovered in the main operating room without going through the post-anesthesia care unit. thirty minutes of room ventilation was performed after tracheal extubation. a physician in the main operating room sent the patient into the corridor, while the other physician outside the main operating room wearing ppe took over and transferred the patient to the general ward directly through the exclusive pathway (fig. 4) . patients moving to the intensive care unit (icu) were transferred while remaining intubated. before transfers, we injected patients with a sufficient amount of intravenous muscle relaxant and sedative drugs to prevent coughing or movement and we applied a portable ventilator or bag-valve mask with a hepa filter to the patient. operations for six mers-related patients. the details of the six mers-related patients undergoing surgery are presented in table 2 . two patients had operations during phase 1 and four patients during phase 2 of the outbreak (fig. 1) . the negative-pressure operating room was set up to be used from phase 2. regarding ppe levels for the hcws attending these six patients, standard ppe was applied during management of patient a (asymptomatic mers-exposed patient), while anesthesiologists wore enhanced ppe for high-risk procedures (tracheal intubation). enhanced ppe was applied to hcws for patient b because the patient was symptomatic and still within the two-week incubation period, even though both pcr results were negative. enhanced ppe, including papr to reduce risk of mers-transmission, was applied for patient c who underwent surgery in the middle of the outbreak (phase 2). papr provides more perfect sealing and protection of the head surface. patients d and e had documented mers-cov infection and their recovery was confirmed with symptom resolution and two negative mers-cov pcr tests. however, enhanced ppe with papr was applied to hcws because the infection risk could not be eliminated during exposure to a large amount of body fluid, especially during cesarean section (patient d). after spinal anesthesia, she recovered in the main operating room and was transferred directly to the general ward. patient f had a history of exposure to a mers patient in the emergency room and was isolated due to a fever. enhanced ppe was applied to the hcws for patient f and she underwent surgery with only one set of negative pcr results because of her emergency condition. mers, as well as sars, is associated with coronaviruses, both of which have high affinity for the lower repiratory tract and easily produce severe pneumonia [21] [22] [23] [24] [25] . although mers has lower human-to-human transmission potential and has resulted in fewer large outbreaks than sars, there may be occasional amplification of clusters in healthcare settings [21] [22] [23] . moreover, mers case fatalities are reported to be much higher than sars (35-45% for mers and 10-15% for sars) 12, 17, 23, 24, 26 . unlike sars, ongoing small and large mers outbreaks in the arabian peninsula foster potential future mers outbreaks in non-endemic countries. however, there is likely to be a very limited number of hospitals that are prepared with negative-pressure operating rooms, except for a few hospitals in hong kong that experienced the 2003 sars outbreak 13 . almost all hospitals generally have positive-pressure operating rooms and they may experience an outbreak without facilities that are prepared for perioperative management of mers patients, as our hospital did in 2015. one of the highlights of our experience during the outbreak was the temporary set-up of a negative-pressure operation room with an adequate pressure gradient (≥2.5 pa) by modifying two connected operating rooms according to us centers for disease control and prevention (cdc) guidelines 15 . continuous negative pressure was maintained in the main operating room (−4.7 pa) and the anteroom (−1.2 pa). this temporary setting was possible because the two adjacent rooms had separate atmospheric air inlets and exhaust systems. although we could not measure the airflow pattern or dispersion of infectious particles directly 27 , the cleanliness levels in both operating rooms were 2,500 particles, well below the institutional target cleanliness for general surgery (<10,000 particles). although the precise route of mers-virus transmission is currently not clearly understood 21 , mers, as well as sars, is known to spread by direct contact with infectious material, such as large respiratory droplets, and also by airborne routes 28, 29 . touching contaminated objects may also be a source of transmission; this is different from tuberculosis, which is transmitted by airborne routes 19, 29 . therefore, when performing procedures that generate aerosols, such as endotracheal intubation, in patients with mers or sars, hcws must wear enhanced ppe, including gloves, a gown, either a face-shield that fully covers the front and sides of the face or goggles, and respiratory protection at least as protective as an n95 filtering face piece respirator 19, 28, 30 . when removing ppe, care should also be taken not to contact contaminated materials. considering potential aerosol generation in operating rooms and the transmission risk of mers-cov while changing ppe, the temporary modification of an operating room to a negative-pressure room with an anteroom should provide suitable protection for hcws participating in operations on mers-related patients 5 . a second highlight of our experience is the highly conservative application of ppe to hcws. at the time of the outbreak, there were no specific guidelines for perioperative management 31 . therefore, we used a conservative approach based on our experience and previous reports. first, although the previous guidelines recommended that asymptomatic mers-exposed patients be managed as general patients undergoing surgery, we applied standard ppe to hcws and we performed mers-cov pcr screening twice. although mers progressed gradually after symptom onset 32 , we could not exclude the possibility that asymptomatic mers-exposed patients had the potential to develop symptomatic disease perioperatively. moreover, we observed development of mers after the known incubation period of 14 days in an immunocompromised host 10, 33 ; thus, the possibility of exceptional cases could be considered. furthermore, a certain proportion of asymptomatic mers-exposed patients could actually be asymptomatic mers-infected patients. approximately 21% of laboratory confirmed mers patients have been classified as asymptomatic or having nonspecific mild symptoms at the time of testing 34 . the potential for transmission from asymptomatic mers-cov pcr-positive person is currently unknown, but there are reports about prolonged viral rna detection in the upper respiratory tract in asymptomatic pcr-positive person 9 . considering these points, it would be reasonable to prepare more conservatively than the existing guidelines call for. www.nature.com/scientificreports www.nature.com/scientificreports/ another point on which our preparations differed from the guidelines was the application of enhanced ppe, which emphasizes full protection of the body surface with a hooded coverall. during the outbreak in our hospital, mers transmission events occurred among hcws who were equipped with standard ppe, including n95 masks. transmission may have occurred after possible contamination of uncovered head or face surfaces 35 . therefore, if a patient with a mers contact history had mers-associated symptoms, applying enhanced ppe (either a n95 respirator or papr) during surgery would be appropriate for hcws because numerous droplets and aerosols may be produced during airway interventions. because the n95 may fit inadequately if worn for a long time or after movement during surgery, wearing papr will be more beneficial. however, unlike hcws dealing with ebola virus, impermeable and fluid-resistant gowns are not required because body fluids are not infectious as with ebola virus diseases 9, 28, 35, 36 . our experience was limited in that, as a mers outbreak outside the endemic country, we did not have an opportunity to perform surgical procedures in actively virus-shedding mers-infected patients. additionally, our infection-prevention protocols would be too conservative to apply in mers-endemic situations. however, considering the potential risk of infected hcws, preventing mers transmission is extremely important in the management of a mers outbreak. importantly, our experience can be generalized to other non-endemic countries for managing potential outbreaks of emerging respiratory diseases. in the era of globalization, a mers outbreak can occur in any country outside the middle east. a very limited number of hospitals are equipped with negative-pressure operating rooms, and therefore, most hospitals are likely to experience a mers or other outbreak in an unprepared circumstance. we hope that this report will help other hospitals in preparing for future mers outbreaks and infection control in unexpected conditions. this study was based on all available data at the samsung medical center from the mers outbreak and on interviews with hcws associated with the outbreak. the study was approved by samsung medical center institutional review board. the documents for review included electronic medical records of mers-related patients who underwent operative procedures and institutional guidelines for perioperative management of mers-related surgical patients. the mers guidelines were prepared through multidisciplinary team discussions that were held by our hospital's infection control department during and after the mers outbreak. the records about the temporary set-up of a negative-pressure operating room were also reviewed. we also collected data through interviews with hcws who participated in surgery and anesthesia for mers-related patients. we defined mers-related patients as those who were possibly or directly exposed to mers or who had a previously confirmed mers diagnosis 10 . in brief, patients who had a potential but unconfirmed close contact history with a mers patient were defined as possibly exposed patients, and they were allowed to continue their normal activities until mers-like symptoms developed. directly exposed patients included those who had close contact with a known mers patient and who did not wear proper ppe; these patients were isolated in their homes or in private negative-pressure rooms at our hospital. because the number of mers-infected patients continuously increased at our hospital, we partially closed the hospital on june 13 10 , at which point outpatient-care clinics were closed and the emergency department was only available for life-threatening emergencies. all elective surgeries were postponed if possible 10 . we defined the early phase of the outbreak (before june 13) as phase 1 and the middle phase of the outbreak (from june 13) as phase 2. for mers-cov pcr tests, either sputum or nasopharyngeal swab samples were collected and sputum samples were preferred if available 14 . sputum was collected directly into a sterile, leak-proof, screw-capped sputum collection sterile container and nasopharyngeal swab was collected with an eswab (482 c, copan diagnostics inc., murrieta, ca, usa). clinical samples were screened by rrt-pcr testing with amplification targeting the upstream e region (upe) and confirmed by subsequent amplification of the open reading frame (orf)1a using powerchek ™ mers real-time pcr kits (kogene biotech, seoul, korea). all rrt-pcr reactions were performed using the 7500 fast real-time pcr system (applied biosystems, foster city, ca, usa). the pcr reaction was performed in a total volume of 20 μl (15 μl pcr reaction mixture and 5 μl template rna). thermocycling conditions included a step at 50 °c for 30 min, followed by 95 °c for 10 min and then 40 cycles of 15 s at 95 °c and 60 s at 60 °c. positive viral template control and no-template control were included in each run. the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was amplified simultaneously as a heterologous internal control to monitor pcr inhibition. a positive test result was defined as a well-defined exponential fluorescence curve that crossed the cycle threshold (ct) < 35 cycles for both upe and orf1a. a sample was considered "equivocal" if the upe result was positive but the ct value for orf1a was >35 and <40. we interpreted the result as "indeterminate" if (1) the upe result was positive but the ct value for orf1a was undetected or if (2) the ct value for upe was >35 and <40. institutional review board statement. the study was performed in accordance with the declaration of helsinki and experimental protocols were revised and approved by irb at samsung medical center. (irb no. smc 2016-08-156-002). informed consent statement. irb at samsung medical center has approved the waiver of patient consent form because of the retrospective nature of this study. patient confidentiality is maintained at all time in accordance with samsung medical center policies. middle east respiratory syndrome: new disease, old lessons isolation of a novel coronavirus from a man with pneumonia in saudi arabia contact investigation for imported case of middle east respiratory syndrome enhanced mers coronavirus surveillance of travelers from the middle east to england laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities family cluster of middle east 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during a hospital-associated outbreak surgical protocol for possible or confirmed ebola cases anesthetic implications of ebola patient management: a review of the literature and policies j.p. this author helped with data collection, data analysis, writing of the first draft and revision of the manuscript, and archiving of the study files. s.y.y. this author helped with data analysis, discussion of results, writing and revision of the manuscript, archiving of the study files and approval of final version. j. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.r.p. or j.j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-016451-k8m2xz0e authors: chertow, daniel s.; kindrachuk, jason title: influenza, measles, sars, mers, and smallpox date: 2020-01-03 journal: highly infectious diseases in critical care doi: 10.1007/978-3-030-33803-9_5 sha: doc_id: 16451 cord_uid: k8m2xz0e influenza, measles, sars, mers, and smallpox illnesses are caused by highly infectious viral pathogens that induce critical illness. these biologically diverse viruses enter and replicate within host cells triggering viraland host-mediated damage that results in pneumonia and multiorgan failure in severe cases. early case identification and strict infection control limit healthcare transmission. vaccination allowed smallpox eradication and limits global measles and seasonal influenza mortality. while sars-coronavirus (cov) is no longer circulating, mers-cov and zoonotic influenza viruses, with pandemic potential, remain persistent threats. supportive critical care is the mainstay of treatment for severe disease due to these viral infections. measles virus is a pleomorphic, enveloped, negative-sense, single-stranded rna virus of family paramyxoviridae of approximately 100 nm to 300 nm in diameter [2] . measles virus causes mild to severe illness during seasonal outbreaks in endemic areas and intermittent outbreaks in nonendemic area [10] . measles virus codes for six structural and two nonstructural proteins (fig. 5 .1b) [11] . hemagglutinin (h) and fusion (f) glycoproteins project from the viral surface and facilitate viral binding to cellular receptors and fusion with the host cell membrane, respectively. matrix (m) protein underlies the envelope providing structure. the inner nucleocapsid is composed of rna coated by nucleoprotein (n), bound by the polymerase complex which includes the large (l) polymerase protein, and phosphoprotein (p), a polymerase cofactor. the remaining nonstructural proteins include c and v. coronaviruses are spherical, enveloped, positive-sense, single-stranded rna viruses of family coronaviridae of approximately 120 nm in diameter [12] . coronaviruses are the causative agents of an estimated 30% of upper and lower respiratory tract infections in humans resulting in rhinitis, pharyngitis, sinusitis, bronchiolitis, and pneumonia [13] . while coronaviruses are often associated with mild disease (e.g., hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1), severe acute respiratory syndrome coronavirus (sars-cov), a lineage b betacoronavirus, and middle east respiratory syndrome coronavirus (mers-cov), a lineage c betacoronavirus, are associated with severe and potentially fatal respiratory infection [14, 15] . sars-and mers-cov transcribe 12 and 9 subgenomic rnas, respectively, which encode for the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural proteins (fig. 5 .1c) [14] . s, e, and m are all integrated into the hostderived lipid envelope, and s facilitates host cell attachment to angiotensinconverting enzyme (ace)-2 receptors for sars-cov and dipeptidyl peptidase (dpp)-4 receptors for mers-cov [16, 17] . the n protein encapsidates the viral genome to form the helical nucleocapsid. the viral replicase-transcriptase complex is made up of 16 nonstructural proteins (nsp1-16) including a unique proofreading exoribonuclease that reduces the accumulation of genome mutations [12] . poxviruses are oval-to-brick-shaped double-stranded dna viruses of family poxviridae that range in size from 200 to 400 nm [2] . viruses within genus orthopoxvirus that cause human disease include cowpox virus (cpxv), monkeypox virus (mpxv), vaccinia virus (vacv), and variola virus (varv), the etiologic agent of smallpox [18] . poxviruses contain a biconcave viral core where the dna genome, dnadependent rna polymerase, and enzymes necessary for particle uncoating reside ( fig. 5.1d ) [19] . this nucleosome is surrounded by a core membrane that is flanked by two proteinaceous lateral bodies. a single lipid membrane surrounds the cellassociated form of the mature virion (mv). a second host-derived lipid envelope covers the extracellular virion (ev) [2, 19] . poxvirus genomes are comprised of a large, linear double-stranded viral dna genome that encodes ~200 genes. highly conserved structural genes are predominantly found in the middle of the genome, whereas variable virulence factor genes that function in immune evasion, virulence, and viral pathogenesis are found at the termini of the genome [20] . wild aquatic birds are natural reservoirs for nearly all influenza a virus subtypes, which spread to domestic avian species and mammals, including humans [5] . h17n10 and h18n11 subtypes are exceptions in that they have only been isolated from bats [6, 7] . certain h5 and h7 subtypes are highly pathogenic to domestic poultry when transmitted from wild birds, known as highly pathogenic avian influenza (hpai) viruses [21] . hpai viruses cause spillover infections in humans that may be severe or fatal. examples include outbreaks of h5n1 and h7n9 hpai viruses in asia with high case fatality among humans, although limited human-tohuman transmission [22, 23] has been reported. hpai virus adaptations might lead to sustained human-to-human transmission, and so poultry outbreaks are managed by flock depopulation [24] . influenza a subtypes isolated in swine include h1 to h5, h9, and n1 and n2. subtypes that spillover into humans cause mild to severe illness and are known as swine "variant" viruses [25] . currently circulating seasonal influenza a subtypes h1n1 and h3n2 and influenza b viruses, yamagata or victoria lineage, cause annual epidemics during fall through spring in temperate regions and infections throughout the year in the tropics [26] . antigenic drift of h and n surface glycoproteins drives annual epidemics. from 2017 to 2018, seasonal influenza caused approximately 49 million illnesses, 1 million hospitalizations, and 79,000 deaths in the united states alone [27] . when two or more influenza a viruses infect a common host, such as a bird or pig, individual gene segments may recombine to form a novel virus, known as antigenic shift. influenza pandemics occur when novel viruses emerge into an immunologically naïve population and become adapted for sustained human-to-human spread. the 1918 "spanish" influenza pandemic was the most severe on record, resulting in an estimated 50 million deaths [28] . less severe pandemics occurred in 1957, 1968, and 2009 . in an effort to improve preparedness and response to seasonal, pandemic, and zoonotic influenza, the world health organization (who) conducts global surveillance of influenza a and b isolates (fig. 5 .2a) [29] . measles is pathogenic for humans and nonhuman primates, although sustained transmission occurs only among humans raising potential for global elimination [30] . historically, measles infected an estimated 90% of children by age 5 years, resulting in approximately 2 million global deaths each year [10] . with the introduction of the measles vaccine in 1963 and advances in global vaccination programs, measles cases and mortality have drastically declined (fig. 5.2b ). by 2017, 85% of children worldwide had received at least one dose of the measles vaccine by age 1 year, and during 2000-2017, global measles mortality decreased by 80%, preventing an estimated 21 million deaths [31] . of the 24 known measles genotypes, only five were detected in circulation during 2016-2017. despite these gains, measles remains endemic in many regions of the world including africa, western pacific, south east asia, and europe, and measles has resurged in previously low-incidence areas (e.g., regions within europe and the americas) with epidemics attributable to importation of cases and suboptimal immunization coverage [32] [33] [34] . an estimated 93% population immunity is required to prevent measles transmission within communities, a prerequisite for global elimination [35] . chinese horseshoe bats are the putative reservoir for sars-cov, and dromedary camels are thought to be the reservoir for mers-cov [36] [37] [38] [39] [40] [41] [42] [43] . animal-to-human transmission likely occurs following direct contact with intermediate hosts [38, 44] . during the 2003-2004 sars epidemic, 8096 cases and 774 deaths were reported from 26 countries with no cases reported since ( fig. 5 .2c) [45] . human-to-human transmission of sars-cov occurred primarily in healthcare settings with healthcare workers comprising 22% and >40% of reported cases in china and canada, respectively [45] . mers was first reported in saudi arabia in 2012 with >2000 cases and >800 deaths reported from 27 countries through 2018 [46] . while most cases have been reported from the arabian peninsula, an imported case to south korea in 2015 resulted in a large outbreak in multiple healthcare facilities [47] . mers transmission occurs primarily in healthcare facilities and to a lesser degree within households [48, 49] . while the only known reservoir for varv is humans, it has been postulated that the virus emerged from an ancestral rodent-borne poxvirus more than 10,000 years ago [18, 50] . numerous smallpox epidemics have occurred throughout recorded history including more than 300 million fatalities during the twentieth century alone [51] [52] [53] . smallpox was eventually eradicated following the implementation of the smallpox eradication program by the who from 1966 to 1980 ( fig. 5 .2d) which was facilitated by the absence of a zoonotic reservoir for varv [51] . influenza viruses are transmitted by large respiratory droplets by coughing, sneezing, or talking or through contact with infected surfaces [54] . influenza viruses bind to sugar moieties on the surface of airway epithelial cells where early viral replication, propagation, and shedding occur during an average 1-2 days of incubation period [55] [56] [57] . peak viral replication typically occurs within 4 days of symptom onset and resolves within 7-10 days, lasting longer in children and immunocompromised hosts [58] [59] [60] . on average one person infects -one to two additional people; however, this reproductive number (r 0 ) varies by viral strain and social and environmental factors [61] . viral infection impairs the airway mucosal barrier and disrupts the alveolar-capillary membrane contributing to leakage of fluid and inflammatory cells into the alveolar space which impairs gas exchange resulting in hypoxemia [62, 63] . bacterial coinfection often complicates severe cases contributing to respiratory failure and death, with staphylococcus aureus and streptococcus species as predominant copathogens [64] . seasonal influenza virus infection is largely limited to the respiratory tract; however, h5 and h7 hpai viruses have a polybasic cleave site within the hemagglutinin allowing for replication outside of the respiratory tract [65, 66] . infection with one strain of influenza does not confer complete immunity to other strains or subtypes [67] . measles is among the most highly contagious respiratory infections, spread by exposure to large respiratory droplets through coughing, sneezing, or talking; by indirect contact with infected surfaces; or by small infectious droplets that can remain suspended in air for up to 2 hours [10, 68] . respiratory tract dendritic cells, lymphocytes, and alveolar macrophages are early targets of infection where during an average 8-to 12-day incubation period measles replicates and spreads to local lymphatics and respiratory epithelium and then disseminates in blood via infected lymphocytes to epithelial and endothelial cells in most organs [69] [70] [71] . the infectious period begins with fever onset and extends for several days after rash appears [72] . the estimated r 0 of measles is 9-18 dependent upon host susceptibility and social and environmental factors [73] . measles infects and disrupts tissues throughout the body; however, severe disease is primarily due to lower respiratory tract and neurological complications [72] . natural measles infection confers lifelong immunity, and passive transfer of maternal antibodies protects newborns during the early postnatal period [74] . individuals who recover from measles infection are at increased risk of secondary infection [75, 76] . sars-cov is transmitted by large respiratory droplets and by contact with infected surfaces. epidemiologic data also support small droplet airborne transmission of sars-cov although the estimated r 0 of 0.86-1.83 argues against this being a predominate route of spread [77, 78] . sars-cov binds to angiotensin-converting enzyme (ace)-2 receptors on respiratory epithelial cells, pneumocytes, and alveolar macrophages resulting in diffuse alveolar damage and respiratory failure [79, 80] . sars is a systemic infection with viremia detected in most cases affecting multiple cell types and organs [81, 82] . acute kidney injury is multifactorial with evidence of acute tubule necrosis, vasculitis, and glomerular fibrosis, and central nervous system manifestations are at least in part attributable to direct infection of neurons resulting in edema and degeneration [83] . mers-cov is transmitted by large respiratory droplets and by contact with infected surfaces with an estimated r0 of <1 to >1 outside of versus within healthcare settings, respectively [84] . mers-cov binds dipeptidyl peptidase 4 (dpp4) on respiratory epithelial cells and pneumocytes where it undergoes productive replication during a 2-14 days incubation period [16] . viral shedding from the lower respiratory tract may persist for weeks [85, 86] . viremia, while not documented in all cases, is associated with severe disease and productive infection of dcs, and macrophages is thought to facilitate immune dysregulation [87, 88] . dpp4 is broadly expressed on cells outside of the lung; however, few autopsy data are available to define viral distribution [16, 89] . varv is transmitted primarily by large respiratory droplets and to a lesser degree through contact with contaminated objects such as scabs, bedding, or clothing or by airborne small respiratory droplets [90, 91] . varv is thought to replicate in airway epithelium and spread to regional lymph nodes [92, 93] . varv replicates within lymph nodes and disseminates via the bloodstream seeding distant sights including skin, spleen, bone marrow, liver, kidney, and other organs [94] . fever manifests following an average 12 days incubation, and rash follows fever by 3-4 days, concurrent with high-level viral shedding from oropharyngeal secretions [95, 96] . the estimated r 0 of smallpox is between 3.5 and 6 [97] . high-level viremia is detected more often with hemorrhagic compared with ordinary type smallpox, although exact mechanisms of organ failure observed in fatal case are not well defined [98] [99] [100] [101] . influenza infection manifests as acute onset of fever, chills, malaise, headache, and myalgias following an average 1-2 days asymptomatic incubation period [9] . most infections are self-limited resolving within 1-2 weeks. upper or lower airway complications include otitis media, sinusitis, bronchitis, and pneumonia with or without bacterial coinfection [63, 64, 102] . risk factors for severe infection include age >65 years or <5 years; pregnancy; preexisting respiratory, cardiac, neurologic, or metabolic conditions; immunosuppression; and obesity. progressive lethargy and shortness of breath, typically within 5 days of symptom onset, suggest development of lower respiratory tract complications which may rapidly progress to respiratory failure and death in severe cases [64] . pneumonia due to influenza infection alone versus influenza and bacterial coinfection cannot be reliably distinguished by clinical or radiological grounds, and so a high index of suspicion is needed. influenza complications outside of the respiratory tract include exacerbation of underlying heart disease including ischemic heart disease and heart failure, myocarditis, encephalopathy, and encephalitis [103] . measles infection manifests by acute onset fever, coryza, conjunctivitis, and cough [10] . small white papules, koplik spots, appear on the buccal mucosa within 3 days of fever onset, followed by development of diffuse maculopapular rash 1 or 2 days later. diarrhea commonly begins shortly following rash onset and may result in dehydration. symptoms typically resolve within 7 days of fever onset in self-limited illness. groups at increased risk for measles complications include malnourished infants and those with vitamin a deficiency, adults >20 years old, and immunocompromised individuals [72] . respiratory complications include otitis media, laryngotracheobronchitis (croup), and pneumonia. pneumonia, often complicated by bacterial coinfection, is the most common severe complication of measles contributing to respiratory failure and death [72, 104] . predominant bacterial copathogens include streptococcus pneumonia, staphylococcus aureus, and haemophilus influenzae. three rare but severe neurologic complications occur [105] . acute disseminated encephalomyelitis (adem) is a demyelinating autoimmune process that occurs within weeks of acute illness in approximately 1 in 1000 cases. adem is characterized by fevers, seizures, and neurologic deficits. measles inclusion body encephalitis is a progressive lethal brain infection occurring within months of acute illness primarily among individuals with impaired cellular immunity. subacute sclerosing panencephalitis (sspe) occurs 5-10 years following initial infection resulting in seizures and cognitive and motor decline resulting in death. sspe affects an estimated 1 in 10,000 infants under 1 year of age and is attributed to host responses to defective viral particle production in the brain. following an average 5-day incubation period, sars-cov infection presents with fevers, chills, dry cough, headache, malaise, and dyspnea commonly followed by watery diarrhea [106] [107] [108] . age >60 years and pregnancy are associated with severe disease manifested by progressive respiratory failure within 2 weeks of illness onset [108, 109] . common laboratory features of sars included lymphopenia, thrombocytopenia, abnormal coagulation parameters, and elevated lactate dehydrogenase, alanine aminotransferase, and creatine kinase levels [110] [111] [112] . acute kidney injury and proteinuria were observed in 7% and 84% of patients, respectively [113] . initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of 5 days [114] . gastrointestinal symptoms, including vomiting and diarrhea, occur in onethird of patients [115] [116] [117] [118] . the median times from symptom onset to hospitalization, icu admission, and death are 4, 5, and 12 days, respectively [118] . mers patients present with a rapidly progressing pneumonia requiring mechanical ventilation and additional organ support with the first week of illness [109] . severe disease has been linked to comorbidities including diabetes mellitus (68%), chronic renal disease (49%), hypertension (34%), chronic cardiac disease (28%), chronic pulmonary disease (26%), and obesity (17%) [114] . the median age of those with confirmed mers is 50 years with a male-to-female ratio of 3.3:1 [114] . laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes [114, 115, 117, 119, 120] . high lactate levels and consumptive coagulopathy have also been reported [119, 121] . chest radiographic abnormalities are due to viral pneumonitis with or without secondary bacterial pneumonia, and acute kidney injury occurs in up to 43% of patients [114, 119, 120, [122] [123] [124] . as the smallpox disease course was related to the clinical presentation of disease, rao proposed a clinical classification system [125] that was later adopted by the who in 1972 [51] . ordinary type smallpox was the most common clinical type of smallpox. the incubation period was 7-19 days and was followed by fever onset (38.5-40.5 °c), headaches, backaches, vomiting, and diarrhea [51] . lesions first appeared on mucous membranes (including the tongue, palate, and pharynx) ~1 day prior to macular rash development, where lesions began on the face followed by proximal regions of the extremities, the trunk, and the distal extremities. lesion development followed a centrifugal dispersion pattern, typically most dense on the face, with papules appearing within 2 days of macular rash development. papules became vesicular ~2-4 days later followed by a pustular stage (5-7 days postrash) that peaked ~10 days postrash. pustule resolution quickly followed and was accompanied by lesion flattening, fluid reabsorption, hardening, and scab formation (14-21 days postrash). rao proposed for ordinary type smallpox to be further subdivided based on the macular rash pattern [125] . these included discrete ordinarytype smallpox, characterized by discrete skin lesions; confluent ordinary-type smallpox, where pustular skin lesions were confluent on the face and extremities; and semiconfluent ordinary-type smallpox, where skin lesions were confluent on the face but disparate over the rest of the body. modified-type smallpox, where lesions were less numerous than in ordinary-type smallpox, was primarily associated with vaccinated individuals and had an accelerated nonfatal disease course [125] . flattype and hemorrhagic-type smallpox were the most lethal forms of the disease but were also very rare (~7% and 3% of patients, respectively) [51] . flat-type smallpox had high cfrs in both unvaccinated and vaccinated patients (97% and 67%, respectively). hemorrhagic-type smallpox was nearly 100% fatal in both vaccinated and unvaccinated individuals, and death normally came prior to macular rash development. the clinical symptoms of flat-type smallpox were more severe during the prodromal period and did not subside. skin lesions were flat and often black or dark purple. respiratory complications were common and patients were febrile throughout disease. death typically occurred 8-12 days post-fever onset. hemorrhagic-type smallpox could be divided into early and late hemorrhagic-type smallpox. the early form was characterized by hemorrhage (primarily subconjunctival) early in the disease course. generalized erythema, petechiae, and ecchymosis within 2 days of fever and flat matter lesions formed across the entire body surface. lesions turned purple by day 4 with death by day 6 as a result of cardiac and pulmonary complications. in the late form, hemorrhages occurred following rash development and death followed between 8 and 10 days post-fever onset. in healthcare settings, patients under evaluation for influenza should be isolated, and standard, droplet, and contact precautions should be implemented [126] . traditional antigen-based rapid diagnostic assays (rdas) for influenza lack sensitivity and cannot be relied upon to rule out infection [26] . newer antigen-based rdas that employ a digital scan of the test strip, and molecular assays that employ isothermal amplification technology have improved sensitivity and specificity that more closely approximates highly sensitive and specific reverse transcriptase polymerase chain reaction (rt-pcr)-based assays [127] . acceptable sample types for influenza testing include nasopharyngeal swab or wash and bronchoalveolar lavage specimens. individuals suspected of zoonotic influenza infection should have case evaluation and specimen testing coordinated through local or state public health authorities. measles should be considered in patients without preexisting immunity and a compatible febrile rash illness. travel to a region with ongoing measles transmission or exposure to other individuals with a febrile rash illness should raise suspicion. patients under evaluation for measles require isolation and implementation of standard, airborne, and contact precautions. local or state health authorities should be contacted within 24 hours to assist with confirmatory testing, case finding, and infection control. measles is typically confirmed by measles-specific igm serology or detection of measles rna in a nasopharyngeal, throat, or urine specimen by rt-pcr [10] . a fourfold or greater rise in measles igg titers between acute and convalescent samples tested 2 or more weeks apart can assist with diagnostic uncertainty. virus can also be cultured from respiratory, blood, and urine specimens in appropriate public health laboratories. while sars is no longer circulating, mers should be suspected in individuals with a compatible febrile illness and an epidemiological risk factor [128] . risk factors include travel to the arabian peninsula or contact with a confirmed or suspected case within 14 days of symptom onset. patients under evaluation for mers require isolation and implementation of standard, airborne, and contact precautions. confirmatory testing and infection control should be coordinated through local or state health authorities. mers may be confirmed in designated public health laboratories by rt-pcr testing of lower respiratory tract specimens [129] . multiple other specimen types including upper respiratory tract samples, serum, and stool should also be collected for testing. serologic testing can be used to evaluate for suspected infection among individuals no longer shedding virus [129, 130] . smallpox has not been observed in over 40 years; however, concerns remain for use as a bioweapon. major and minor criteria have been established to assist clinicians in recognition of smallpox [131] . individuals under evaluation should be isolated, and standard, airborne, and contact precautions should be implemented. local or state health authorities should be contacted to assist with confirmatory testing and public health interventions. pcr identification of variola dna or isolation of the virus from a clinical specimen is required to confirm a diagnosis in specialized highcontainment laboratories. annual seasonal influenza vaccination is recommended in the united states for all individuals aged 6 months or older and has been associated with decreased risk of pneumonia and death, particularly among high-risk groups [132] [133] [134] . seasonal influenza vaccination does not provide protection against novel strains. consequently, efforts are underway to develop a vaccine that would protect against most or all influenza strains [135] . three classes of drugs are licensed for the treatment of influenza in the united states [136] . adamantanes, including amantadine and rimantadine, are not currently recommended given resistance of circulating seasonal strains. baloxavir morboxil, a cap-dependent endonuclease inhibitor, was recently approved for the treatment of uncomplicated influenza [137] . neuraminidase inhibitors (nai) include oral oseltamivir, inhaled zanamivir, and intravenous peramivir. prophylactic use of nais is recommended in unvaccinated individuals with risk factors for severe disease and during institutional outbreaks to limit spread. therapeutic use is recommended for individuals with suspected or confirmed influenza that have developed or are at high risk for influenza complications [26] . influenza complications, including respiratory and multiorgan failure, are managed with supportive care. bacterial coinfection should be considered and empirically treated early pending results of microbiologic testing among severe cases. measles can be effectively prevented through vaccination, typically given in combination with vaccines for rubella (mr), mumps (mmr), or varicella (mmr-v). who recommends the first dose of measles vaccine be administered at 9 or 12 months of age in high and low prevalence settings, respectively [138] . a second dose should be administered after a minimum of 4-week interval. nonimmune individuals that have been exposed to measles should receive post-exposure prophylaxis with mmr or immunoglobulin within 72 hours or 6 days, respectively, although not concurrently [139] . clinical management of patients with measles consists of fluid, electrolyte, and nutritional support and early recognition and treatment of bacterial coinfection [10] . two doses of vitamin a in children under 2 years have been associated with reduced risk of pneumonia and death [140] . who recommends administering 200,000 iu of vitamin a daily for 2 days in children aged 1 year and older, with reduced dosing in younger infants [141] . there are currently no licensed therapeutics or vaccines for sars or mers. consequently, supportive care is the mainstay of treatment [142] . renal replacement therapy is frequently required in severe illness [119, 143, 144] . empiric antibiotics are often administered given potential for secondary bacterial infection. ribavirin and pegylated interferon alpha 2b have been administered to mers patients, although effectiveness data is lacking [144] . aerosol-generating procedures including endotracheal intubation are associated with increased risk of healthcare worker infection necessitating strict adherence to infection control measures, including use of eye protection in addition to standard, airborne, and contact precautions [145] 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syndrome coronavirus infection: a single-center experience in saudi arabia bombay: the kothari book depot prevention strategies for seasonal influenza in healthcare settings diagnostic accuracy of novel and traditional rapid tests for influenza infection compared with reverse transcriptase polymerase chain reaction: a systematic review and meta-analysis interim patient under investigation (pui) guidance and case definitions respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome epidemiology of coronavirus-associated respiratory tract infections and the role of rapid diagnostic tests: a prospective study evaluating patients for smallpox: acute, generalized vesicular or pustular rash illness protocol effectiveness of influenza vaccination in institutionalized older adults: a systematic review university of nottingham influenza and the immunocompromised (uniic) study group, nguyen-van-tam js. influenza vaccination for immunocompromised patients: systematic review and meta-analysis by etiology prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states the pathway to a universal influenza vaccine influenza antiviral medications: summary for cli baloxavir marboxil investigators g. baloxavir marboxil for uncomplicated influenza in adults and adolescents world health organization. measles vaccines: who position paper measles (rubeola): for healthcare professionals vitamin a for treating measles in children middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease recovery from severe novel coronavirus infection ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review smallpox vaccine and its stockpile in 2005 clinical guidance for smallpox vaccine use in a postevent vaccination program the efficacy of vaccinial immune globulin. a 15-year study experience of anti-vaccinia immunoglobulin in the united kingdom efficacy of tecovirimat (st-246) in nonhuman primates infected with variola virus (smallpox) an orally bioavailable antipoxvirus compound (st-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge key: cord-305317-08a1oin2 authors: maltezou, helena c.; tsiodras, sotirios title: middle east respiratory syndrome coronavirus: implications for health care facilities date: 2014-12-31 journal: american journal of infection control doi: 10.1016/j.ajic.2014.06.019 sha: doc_id: 305317 cord_uid: 08a1oin2 background middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus that causes a severe respiratory disease with high case fatality rate. starting in march 2014, a dramatic increase of cases has occurred in the arabian peninsula, many of which were acquired in health care settings. as of may 9, 2014, 536 laboratory-confirmed cases and 145 deaths have been reported globally. methods review of publicly available data about mers-cov health care–associated transmission. results we identified 11 events of possible or confirmed health care–associated transmission with high morbidity and mortality, mainly among patients with comorbidities. health care workers are also frequently affected; however, they tend to have milder symptoms and better prognosis. gaps in infection control were noted in all events. currently, health care–associated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic in countries in the arabian peninsula. conclusion there is a need to increase infection control capacity in affected areas and areas at increased risk of being affected to prevent transmission in health care settings. vaccines and antiviral agents are urgently needed. overall, our knowledge about the epidemiologic characteristics of mers-cov that impact health care transmission is very limited. as the mers-cov epidemic continues to evolve, issues concerning best infection control measures will arise, and studies to better define their effectiveness in real life are needed. middle east respiratory syndrome coronavirus (mers-cov) is a novel betacoronavirus of the coronaviridae family that causes a severe respiratory disease with a high case fatality rate. [1] [2] [3] [4] the virus was isolated for the first time in september 2012 from a 60-year-old patient with fatal pneumonia in saudi arabia. 5 however, the earliest identified human cases were traced back to march 2012, to a cluster of severe respiratory infections in a hospital in jordan. 6 up until now, all mers-cov infected cases are directly or indirectly linked to the middle east; therefore, the name mers-cov was established. 4 over the first 2 years after the emergence of mers-cov, the world health organization (who) has been notified of 191 laboratory-confirmed cases, of which 82 were fatal. 7 however, starting in mid-to late march 2014, a dramatic increase of cases has been recorded, many which were acquired in health care settings and concerned health care workers (hcws). 8 as of may 9, 2014, 536 laboratory-confirmed cases and 145 deaths have been reported to the who globally. 8 as a result, concerns have been expressed about the possibility of a virus genetic change conferring increased transmissibility, and the novel virus received media attention globally. in the context of uncertainties about its epidemiology, the high case fatality rate, the urgent need for a specific antiviral treatment, and the unavailability of a vaccine, mers-cov has been a major public health concern of global dimensions. given the current local epidemiologic trends of mers-cov 8 and the large numbers of travelers that fly out of the arabian peninsula, 9 it is almost certain that an increasing number of cases will be exported to other countries; these cases, especially when that patient is seriously ill, will require medical attention and hospitalization. herein, we review publicly available data about mers-cov focusing on health careeassociated transmission. aspects relevant to infection control are also discussed. we searched pubmed from september 2012 through june 19, 2014, using the terms middle east respiratory syndrome, mers, and novel coronavirus. the abstracts of articles identified through the first pubmed search were screened, and articles presenting original data on health careeassociated infections and outbreaks were included. the reference lists of these articles were also reviewed as were any relevant review articles. in addition, we searched the web sites of the who, united states centers for disease control and prevention (cdc), and european centre for disease prevention and control (ecdc). in total, we reviewed 252 articles on mers-cov and identified 10 articles presenting original data about 11 possible or confirmed health careeassociated transmission events. details about the health careeassociated transmission ranged widely among these articles. to the best of our efforts, we avoided presenting duplicated data. in addition, we selected 30 original and review articles. all articles were studied by both authors independently. mers-cov infection so far has been described in 10 countries in the middle east (saudi arabia, united arab emirates, qatar, jordan, oman, kuwait, egypt, yemen, lebanon, iran), 6 countries in europe (united kingdom, germany, france, italy, greece, the netherlands), 2 country in africa (tunisia, algeria), 2 countries in asia (malaysia, the philippines), and 1 county in the americas (united states). 10 molecular analyses of mers-cov or similar viruses from bats and camels suggest that these 2 species are the natural reservoirs of the virus. 11, 12 whole genome sequencing showed that human and camel viruses from saudi arabia are indistinguishable. 12 multiple transmission routes are suspected; however, their exact contribution has not been elucidated so far. a phylogenetic study of 21 mers-cov genomes from saudi arabia suggested that both humanto-human transmission and sporadic zoonotic events occur. 13 the stability of the virus for prolonged periods in camel milk suggests the potential of excretion of the virus into camel milk and spread through consuming raw milk. 14 the upsurge of cases since mid-march in the arabian peninsula (mainly in saudi arabia) is possibly attributed to an increase in the number of primary cases and hospital-acquired cases, some as a part of mainly small (1-2 cases), but in a few instances large, outbreaks. 8 a change in the transmissibility pattern of the virus and increased efficacy for sustained transmission could facilitate inhospital transmission; however, epidemiologic and molecular data to this effect do not exist. family clusters of mers-cov have been recorded. 15 the secondary attack rate in families was 1.35% in saudi arabia in 2014. 8 among imported travel-associated cases, very few instances of person-to-person transmission have been verified. [16] [17] [18] recent phylogenetic analysis using human sequences from jeddah suggests that the virus has not changed from previous strains. 8 overall, it seems unlikely that the virus has increased its transmissibility or patterns of transmission. the basic reproductive number has been estimated to be <1 using real-time data until june and august 2013, respectively, 19,20 even though the upper range of estimates exceeded 1 in a scenario where infection control was not implemented. 20 these findings indicate no pandemic potential for mers-cov so far. recently, a committee appointed by the who concluded that the conditions for a public health emergency of international concern have not yet been met. moreover, increased testing rates of less ill or asymptomatic cases may have contributed to the upsurge of detected cases. regarding characteristics of affected patients, most are men (male-to-female ratio: 2:1), with a median age of 49 years (range, 9 months-94 years). 8 the spectrum of mers-cov infections ranges from asymptomatic infection to very severe pneumonia with acute respiratory distress syndrome, septic shock, and multiorgan failure resulting in death. in an analysis of 144 confirmed and 17 possible cases, symptomatic patients typically had fever and cough, chills, sore throat, myalgia, and arthralgia, whereas vomiting and diarrhea were present in at least one third of patients. 21 in the same study, 63.4% of patients developed severe respiratory disease. it appears that severe disease predominantly occurs in patients with comorbidities; 76% of the patients in this report had at least 1. 21 the overall case fatality with the latest who figures is 27%. 8 from the very first events of the mers-cov epidemic, the virus showed its health careeassociated dynamic. 6 apart of sporadic community cases and family clusters, health careeassociated transmission has been reported on several occasions during the last 2 years, indicating human-to-human, although inconsistent, transmission (table 1) . 2, 3, 6, 8, [22] [23] [24] [25] [26] [27] gaps in infection control were the common denominator in the events of health care associatede transmission. 2, 3, 6, 8, 22, 24 during the largest so farepublished outbreak of mers-cov that occurred in al-hasa, saudi arabia, in 2013, 4 health care facilities were affected through transfer of patients but also possibly because of repeated introductions of cases from the community. 3 the outbreak extended for almost 2 months and involved 34 cases, including 2 hcws. most cases were confined in the hemodialysis unit with rapid transmission and high attack rates. 3 this outbreak gave the opportunity to elucidate several epidemiologic parameters of secondary mers-cov infection, such as the incubation period (5.2 days; 95% confidence interval, 1.92-14.7 days), serial interval (7.6 days; 95% confidence interval, 2.5-23.1 days), and heterogeneity in transmission, with many infected patients not transmitting the infection at all and 1 infected patient transmitting the infection to 7 others. 3 moreover, this outbreak raised the possibility of transmission through direct or indirect contact and between rooms in the same ward. 3 a recent study showed that mers-cov remained viable for up to 48 hours under specific environmental conditions, which mimic the hospital environment (20 c with 40% relative humidity), whereas its stability was not reduced during aerosolization. 28 these data show that mers-cov has the potential to spread through contact or fomites caused by prolonged survival. a model-based study found that the virus structural characteristics render it very likely to remain viable in the environment for a long period and support fecal-oral transmission. 29 vomiting and diarrhea are common in patients with mers-cov 1,22,27 and may contribute to transmission. the mers-cov case imported in france shared his bathroom with the secondary hospital-acquired case, which raises the possibility of spread through stools. 22 mers-cov is predominantly shed through respiratory secretions during cough. mers-cov has been detected through polymerase chain reaction for up to 16 days in respiratory specimens and stools and up to 13 days in urine. 22, 30, 31 our knowledge about virus shedding and viral load kinetics throughout the clinical course of ill patients is scarce and therefore can provide limited guidance about the duration of implementation of infection control measures. 31 the possibility of prolonged shedding under an immunocompromised status should also be investigated and considered for infection control purposes. health careeassociated mers-cov infections and outbreaks have been associated with high morbidity, high rates and prolonged use of mechanical ventilation, and fatality rates up to 65%. 2, 3, 23 given the fact that health care services are often used by older people with comorbidities and in association with the severe course in the description of demographics of secondary mers-cov cases, a drop of the median age from 59 to 43 years old compared with primary cases has been reported. 21 this depends on the conditions of each outbreak and may be affected by the preponderance of affected hcws in each instance. for example, in the most recent who report, 8 the hcws who tested positive for mers-cov in the 2014 jeddah outbreaks were more likely to be younger, women, and to exhibit mild or no symptoms compared with primary cases. however, 15% of hcws developed a severe disease, which resulted in admission to an intensive care unit or death. 8 unsuspected cases are the main source for the introduction of mers-cov virus from the community or another health care facility. 3 although such patients may present with compatible symptoms, the diagnosis may not be considered early or symptoms may be mild. 2, 24, 33 in the hospital outbreak that occurred in saudi arabia in 2013, 3 patients exhibited no fever during initial presentation. 3 hcws may acquire mers-cov infection either in the community or through occupational exposure. 2, 3, 32 nurses are mostly affected, which is attributed to their prolonged, repeated, and closer physical contact with patients. hcws may continue working despite being symptomatic. 6 an asymptomatic or mildly symptomatic course has been described in hcws, 2, 8, 24 which raises the possibility of transmission of the infection to their vulnerable patients during an asymptomatic phase or early incubation. patient-to-patient transmission has been noted as well. 3, 22 currently, health careeassociated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic. 8 in the recent mission report by the who authorities evaluating data on 128 laboratory-confirmed cases in 14 hospitals in jeddah, saudi arabia, with onset of symptoms between february 17 and april 26, 2014, one-third of the cases were considered to be primary cases (some of the investigations are still ongoing), whereas >60% of the cases (including 39 hcws) were classified as hospital acquired. 8 in the rest of saudi arabia, 26 out of 127 (20.5%) recent cases were identified in hcws. 8 overall, 65 of the 290 (22.4%) cases reported from saudi arabia from march 27 to may 9, 2014, were hcws. 23 in mecca, another large outbreak in a hospital was described with 28 laboratory-confirmed cases, including 27 hcws. 8 both outbreaks were larger than the originally described outbreak in saudi arabia. 3 in the united arab emirates, hcws accounted for more than twothirds of 37 cases reported during the same period. 8 although the who points to infection control gaps for the recent propagation of mers-cov within health care facilities in saudi arabia and the united arab emirates, we do not know if this concerns the use of personal protective equipment, hand hygiene, procedures, environmental cleaning, or triage. given that no vaccines or specific antiviral prophylaxis against mers-cov are available, 35,36 the prevention and control of transmission of mers-cov within health care facilities relies solely on early detection, isolation, and strict implementation of infection control measures. rapid and accurate diagnosis is crucial to trigger contact tracing in the hospital and the community and should be ordered as soon as possible in the context of a relevant epidemiologic profile but also in the event of a health careeassociated cluster of severe respiratory illness cases. patients with confirmed or suspected mers-cov infection should be cared under contact and droplet precautions until testing results. in accordance with who guidelines, a high protection mask (eg, n95 respirator) along with eye goggles, gowns, and gloves should be used during aerosol-generating procedures; the latter should be performed in an adequately ventilated room (minimum of 6-12 air changes per hour) (airborne infection isolation room). 37 for consistency with the recommendations during the 2009 h1n1 pandemic, the united states cdc recommends the use of n95 respirators in all contacts with a laboratory-confirmed or suspected mers-cov infected case. 38 the rationale for this recommendation relies on the gaps of knowledge about the potential for airborne transmission of the novel coronavirus. however, n95 respirators are less tolerated by hcws and are more expensive. 39 the united states cdc also recommends that patients with confirmed or suspected mers-cov infection are placed in an airborne infection isolation room. 38 hcws with mers-cov infection should be strictly excluded from patient care, even with mild symptoms. the role of asymptomatic hcws is under question. overall, there is a need to increase infection control capacity in affected areas and areas at increased risk of being affected to prevent transmission in health care settings. our knowledge about the epidemiologic characteristics of mers-cov that impact health care transmission is very limited. to interrupt in-hospital transmission, routes of efficient exposure and virus shedding should be well studied. the contribution of primary cases to the so-called hospital-acquired cases in the recent upsurge of detected cases in the arabian peninsula is still unclear, and further epidemiologic data and analyses are necessary. in 1 analysis, 60 of 95 (63.2%) cases with evidence of secondary transmission acquired the infection in the hospital environment; nevertheless, 49 of them had additionally reported exposure to animals, therefore not eliminating an alternative source of infection. 21 the stability of the proportion of asymptomatic versus symptomatic cases is an argument against increasing testing as a possible explanation for either primary or secondary cases. 10 on the other hand, a reverse scenario could be that additional cases are missed because cases at the early incubation period or with low viral loads may be missed with molecular testing. a transmission event under similar circumstances has been described in the community for the first imported mers-cov case in the united states that tested negative by molecular assays but subsequently tested positive by serology. 25, 40 research for the future active surveillance and testing are of outmost importance to provide answers about the epidemiology of mers-cov and evolution of the current epidemic. case-control, serologic studies in exposed hcws are needed to better define the effectiveness of infection control measures. transmission of the virus via asymptomatic shedding in feces or other routes (eg, fomites, environment) is another topic for investigation. studies of viral kinetics in affected patients with molecular analyses of samples from various body sites will provide answers for infection control as well. a vaccine against mers-cov should be developed along with specific antiviral agents. there is no doubt that mers-cov remains a serious threat and has exhibited a significant public health impact in the affected countries. currently, health careeassociated transmission plays a pivotal role in the evolution of the mers-cov epidemic in countries in the arabian peninsula. a significant cost has been encountered in terms of personnel and time required for contact tracing and means of implementing infection control and prevention measures in health care settings. so far, there is no evidence of sustained humanto-human transmission. however, significant concerns exist in terms of the increased number of health careeassociated cases, gaps in knowledge regarding transmission routes, and limited infection control capacity in affected countries. as the mers-cov epidemic continues to evolve, vaccine and specific antiviral agents against mers-cov are urgently needed. studies about the effectiveness of infection control measures will provide answers and eventually promote safety in health care facilities both for patients and hcws. epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory 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syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study stability of middle east respiratory syndrome coronavirus in milk family cluster of middle east respiratory syndrome coronavirus infections investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov). ninth update interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case middle east respiratory syndrome coronavirus infections in health care workers first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description a case of imported middle east respiratory syndrome coronavirus infection and public health response stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions prediction of intrinsic disorder in mers-cov/ hcov-emc supports a high oral-fecal transmission severe respiratory illness caused by a novel coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study contact investigation of a case of human novel coronavirus infection treated in a german hospital current advancements and potential strategies in the development of mers-cov vaccines broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) preventing the spread of influenza a h1n1 2009 to health-care workers illinois resident who had contact with indiana mers patient tests positive for mers coronavirus key: cord-275313-mfyff9ne authors: modjarrad, kayvon title: treatment strategies for middle east respiratory syndrome coronavirus date: 2016-01-01 journal: journal of virus eradication doi: nan sha: doc_id: 275313 cord_uid: mfyff9ne middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than 1600 people, resulting in almost 600 deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of this novel pathogen have created an urgent need for effective public health countermeasures, including safe and effective treatment strategies. despite the relatively few numbers of cases to date, research and development of mers-cov therapeutic candidates is advancing quickly. this review surveys the landscape of these efforts and assesses their potential for use in affected populations. respiratory tract infections are the leading cause of mortality in resource-limited settings, accounting for more than 4 million deaths each year globally [1] . epidemic-and pandemic-prone respiratory viruses are the aetiological pathogens in many cases, and have caused several of the most prominent infectious disease outbreaks of the past two decades: these include h5n1 influenza in 1997, severe acute respiratory syndrome (sars) in 2003 and pandemic h1n1 influenza in 2009. most recently, middle east respiratory syndrome coronavirus (mers-cov) has emerged as a novel cause of severe acute respiratory illness after first being identified in a saudi arabian patient in 2012 [2] . although initially restricted to the arabian peninsula, this emerging pathogen has respectively infected and killed more than 1600 and 580 people on four continents across 26 countries [3, 4] . phylogenetically related to sars-cov [5] , mers-cov has a similar clinical presentation [6] [7] [8] [9] , albeit with a higher case fatality rate (~40% versus 10%) [3] [4] [5] . dromedary camels serve as the principal animal reservoir for this virus; and zoonotic spillover from dromedaries to humans has, thus far, driven the course of the epidemic [10] [11] [12] [13] [14] [15] [16] [17] [18] . although humanto-human transmission has been documented -particularly in the context of nosocomial outbreaks [19] [20] [21] [22] [23] [24] -the spread of mers-cov is inefficient and unsustained, as reflected in an estimated reproduction rate of no higher than 0.7 [25, 26] . mers-cov is an enveloped, single-stranded, positive-sense rna virus that comprises a 30-kilobase genome that codes for four structural proteins and an rna polymerase [27] , typical of the coronaviridae family ( figure 1 ). the most immunogenic of these proteins is the virus' only surface glycoprotein, spike (s) [28] [29] [30] that mediates viral attachment and fusion via the host cognate receptor, dipeptidyl peptidase 4 (dpp4) [31] . although the broad principles of the virus' life cycle and its mechanisms of pathogenesis are beginning to be understood, this knowledge has not yet translated to a licensed therapy or vaccine. much of the work to develop safe and effective mers-cov countermeasures has centred on vaccines, but the relatively low prevalence of the disease, the sporadic nature of the case clusters and the dearth of detailed knowledge on chains of transmission highlight the need for greater investments into the discovery of effective therapeutic and secondary prophylactic regimens for infected and exposed individuals. efforts to research and develop treatment strategies for mers-cov are accelerating but remain limited in their scope and stage of advancement. there are few novel compounds being studied that are specific for mers-cov molecular targets, as most treatment options, investigational and licensed, are being repurposed from their use for other rna viruses or other non-infectious diseases. the current landscape of mers-cov therapies, therefore, is dominated by an armamentarium of repositioned drugs with in vitro activity against mers-cov replication, but is also speckled with agents that are directed towards and derived from host immunity. the current review surveys the landscape of therapeutic products in each category and assesses their potential for advanced testing and development. respiratory and circulatory support, preservation of renal, hepatic and neurological function, and prevention of secondary infections. beyond implementing basic principles of critical care medicine, immune-based therapies have been used most commonly during both the sars-cov pandemic of 2003 and the current mers-cov epidemic, each time yielding equivocal results. there have been some promising animal data where combination treatment with ribavirin and interferon (ifn)-α2b improved clinical outcomes in mers-cov-infected non-human primates (nhps). however, treatment was initiated very soon after viral challenge (~8 hours), a window that is unlikely to be replicated in a real-world clinical setting [32] . various ifn regimens, in combination with ribavirin, have been intermittently administered to severely ill patients, although typically in an ad hoc manner and in the absence of systematic evaluation [33] [34] [35] [36] [37] . individual case reports and uncontrolled case series not only limit determination of whether an intervention works but if it is safe as well. ribavirin, for example, is a potent nucleoside analogue that has been used with varying measures of success against a range of rna viruses [38] . however, patients can experience significant toxicities when given the drug alone or in combination with an interferon, including but not limited to haemolytic anaemia and metabolic abnormalities. interferons also can elicit systemic adverse effects, psychiatric disturbances and neutropenia [39] . thus, without the benefit of randomised controlled trial data, it becomes difficult to assess whether the treatment is worse than the disease. certain strategies, however, have been shown to worsen clinical outcomes in the setting of a coronavirus infection. for example, studies during the sars pandemic showed that corticosteroids, when used early on sars-cov infected patients, significantly increased viral load, icu admission and mortality [40, 41] . the role for interferon therapies has been less clear in the current mers-cov epidemic, as some data show a positive impact on proximate outcomes, such as oxygenation and inflammation, but no effect on more significant outcomes like hospital stay and long-term survival [35, 36, 42] . rapidly scaled treatments based on naturally occurring neutralising antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have been shown to be relatively safe and potentially effective for reducing mortality from several infections such as sars-cov and influenza [43] [44] [45] , and may hold promise for mers-cov as well. this strategy, however, relies on the rapid identification of cases and contacts and immediate deployment of products to have maximal impact. one study found that convalescent plasma decreased mortality in sars-cov patients only if administered within 14 days of illness [44] . a network for the use of convalescent plasma for case clusters of mers-cov is currently being assembled [43] to test its safety, efficacy and feasibility. however, actualisation of this plan is limited by logistical challenges, local technical capacity and donor supply. unfortunately, no host-derived experimental interventions have yet demonstrated appreciable benefit in acutely ill, mers-covinfected patients in a consistent or controlled manner. this reality, although, has not slowed down the discovery and advancement of passive prophylactic products derived from vaccinated and infected animals and humans. despite intensive efforts to develop a mers-cov vaccine, the prevalence and transmissibility of this emerging pathogen are both relatively low [3, 26] , making it difficult to define a target population for vaccination. mabs, on the other hand, can be administered in the setting of an outbreak without the need to discriminate who might be at greatest risk for infection. they can be used to treat cases early in their natural history and for post-exposure prophylaxis of case contacts. mabs also carry the benefits of higher potency, greater specificity, more extensive pre-licensing evaluation and consequently a more vetted safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic-level detail for the design of better immunogens. they also have been proven as effective therapies in the areas of cancer treatment and autoimmune disease management. although there is only one pathogen, respiratory syncytial virus, for which a mab is licensed for use, there are a number of other infectious disease indications-such as ebola virus disease treatment and human immunodeficiency virus primary and secondary prevention-for which mabs are being tested in advanced phase clinical trials (www.clinicaltrials.gov). despite all of these advantages, the timelines and costs of mab research and development (r&d) are respectively longer and higher than that for polyclonal antibody preparations. in spite of the requirements for greater upfront investments and a more rigorous testing and approval process, several groups have identified highly potent mers-cov mabs and are advancing them through preclinical stages of development (table 1) . some have been isolated from immunised animals (mice/humanised mice/nhps) [46] [47] [48] [49] [50] [51] [52] [53] [54] , while others have been identified from either an antibody human phage library [55] or memory b cells of infected and recovered human survivors [56] . almost all of the published mabs and all of those in development target the s receptor-binding domain (rbd), which contains the most immunogenic epitopes on the virus. many bind to the rbd, expressed both on a recombinant s and on the surface of live virus, with picomolar affinity and neutralise mers-cov at a half maximal inhibitory concentration (ic 50) of 10 ng/μl or less. additionally, several groups have demonstrated new york blood center mersmab1 s1 imunised mouse rbd in vitro [46] nih national cancer institute m336, m337, m338 human antibody library rbd in vitro [52] nih niaid d12, f11, g2, g4 s/s1 immunised mouse rbd, s1, s2 nhp efficacy [51] regeneron regn3048/regn3051 humanised mouse rbd mouse/nhp efficacy [49] tsinghua university mers-4, mers-27 human antibody library rbd in vitro [47] rbd: receptor binding domain; s: spike glycoprotein; s1: spike domain containing rbd; s2: spike domain containing fusion machinery. journal of virus eradication 2016; 2: 1-4 protective efficacy in pre-and post-exposure prophylaxis animal models. because most of the antibodies target the rbd, there is a potential for viral escape from any one mab. thus, there may be a need to develop antibodies against other vulnerable sites on s or to investigate the use of combination mabs to overcome the potential emergence of therapeutic resistance. it is likely that mabs directed at other sites on the s glycoprotein have already been recovered but are not as potent neutralisers, as is the case in one report [51] . a more efficient search for potent neutralising antibodies that target epitopes outside the rbd could be facilitated by a more detailed understanding of the atomic-level structure of the entire s glycoprotein, as has already been resolved for the rbd. the successes thus far in isolating potent and protective mabs, although significant, are likely to be tempered by the challenges of advancing these products to licensing and full-scale production at affordable costs for as of yet undefined populations in a relatively short timeframe. thus, mabs should be advanced along a development pipeline in parallel with a program of rational drug design and discovery. although intensive, supportive care still serves as the primary treatment option for mers-cov and mabs are the focus of the most advanced r&d efforts, antiviral therapies are being actively investigated for use in severely ill patients. there are two main pathways for drug discovery: de novo development and repurposing licensed medications. there are few new antivirals for mers-cov; however, the ebola epidemic has had an unanticipated consequence of facilitating their development. one in particular, gs-5734 developed by gilead sciences, is an adenine analogue that is incorporated into viral rna to disrupt replication [57] . it has shown survival benefit in nhps inoculated with ebola virus and is now advancing through a phase i dose escalation trial. it has been claimed to have in vitro activity against mers-cov as well, but publication of these data is pending. similarly, bcx4430 is a nucleoside analogue that is being developed by biocryst pharmaceuticals for potential treatment of filoviruses, coronaviruses and other rna viruses [58] . additionally, small interfering rna molecules and peptide inhibitors are being investigated for their ability to disrupt mers-cov replication, although these products are still in very early phases of investigation [59, 60] . as the life cycle and genetic sequence of this new coronavirus has become better elucidated, the rational design and development of novel and approved agents with potent antiviral activity have become possible. the advent of high-throughput screens of licensed compounds and small molecules has also allowed researchers to efficiently evaluate large libraries of drugs for their in vitro antiviral activity against novel targets [61] [62] [63] [64] [65] [66] . to date, several dozen licensed drugs have been reported to inhibit mers-cov replication. using slightly different screening technologies, different groups have converged on some common classes of compounds, including nucleoside analogues, antibacterial protein synthesis inhibitors, kinase signalling modifiers, antimetabolites and antiprotozoal agents. mycophenolic acid, an inhibitor of both t an b lymphocytes, has also been found to have strong activity against mers-cov, as it does against other rna viruses such as west nile, hepatitis c and dengue [63] . only one of the drugs to show in vitro activity against mers-cov, lopinavir, however, has been tested in an animal model. mers-cov-challenged marmosets that were treated with this protease inhibitor had better clinical, pathological, virological and radiological outcomes than controls or those treated with mycophenolate mofetil [67] . additionally, two peptides, hr1p and hr2p are being developed as potential fusion inhibitors [59] . by acting on the six-helix bundle core of the mers-cov s protein to prevent protein-mediated cell-to-cell fusion, this class of compounds may hold potential beyond mers-cov towards a long-term objective of a pan-coronavirus antiviral. given some of the common life cycles and pathways of pathogenesis for rna viruses and homologies in protein structures across different coronaviruses, there may be economies of effort and investment in developing antivirals that have activity against more than one virus or family of viruses. irrespective of the breadth of these novel or repurposed compounds, treatment studies should be carried out prospectively according to protocols that plan for the collection of quality controlled data and serial biological sampling to assess viral evolution and biomarkers of favourable clinical outcomes. recent infectious disease outbreaks such as the 2009 h1n1 influenza pandemic, the h7n9 influenza epidemic in china, the ebola crisis in west africa and now the mers-cov outbreak have highlighted the need for better r&d preparedness and improved coordination of clinical testing in the face of the accelerating number of emerging and re-emerging infectious diseases. the ability to have an armamentarium of countermeasures and clinical trial infrastructure in the early phases of an outbreak is critical for mounting an effective public health campaign. for example, the sars-cov pandemic caused more than 8000 cases of severe acute respiratory illness and nearly 900 deaths but few prospective, controlled studies were undertaken to determine the optimal management of the disease. consequently, treatment options for sars-cov were never defined clearly and thus difficult to adapt for mers-cov. although global coordination has resulted in the advancement of some urgently needed, novel countermeasures for mers-cov, they will have to be developed along faster timelines than before, with greater investments earlier in the preclinical development pipeline that can generate products for more timely efficacy testing in affected populations. as the global community takes lessons from the most recent outbreak and prepares for the potential of another regional epidemic or broader pandemic, stakeholders in mers-cov r&d must set out a sound strategy now for where to best target their investments in anticipation of the changing dynamics of the current and future outbreaks. global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of 11 european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov) from sars to mers: 10 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treat emerging infectious diseases: focus on ebola virus disease a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for middle east respiratory syndrome coronavirus (mers-cov) evaluation of candidate vaccine approaches for mers-cov exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus nucleotide prodrug gs-5734 is a broad-spectrum filovirus inhibitor that providescomplete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx4430 in a non-human primate model of filovirus infection structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus design of potential rnai (mirna and sirna) molecules for middle east respiratory syndrome coronavirus (mers-cov) gene silencing by computational method antiviral drugs specific for coronaviruses in preclinical development a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors alternative screening approaches for discovery of middle east respiratory syndrome coronavirus inhibitors treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. key: cord-318181-xxc7vdnt authors: ahmed, anwar e.; al-jahdali, hamdan; alshukairi, abeer n.; alaqeel, mody; siddiq, salma s.; alsaab, hanan; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia date: 2018-03-14 journal: int j infect dis doi: 10.1016/j.ijid.2018.03.005 sha: doc_id: 318181 cord_uid: xxc7vdnt background: the rapid and accurate identification of individuals who are at high risk of middle east respiratory syndrome coronavirus (mers-cov) infection remains a major challenge for the medical and scientific communities. the aim of this study was to develop and validate a risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. methods: a two-center, retrospective case–control study was performed. a total of 360 patients with confirmed pneumonia who were evaluated for mers-cov infection by real-time reverse transcription polymerase chain reaction (rrt-pcr) between september 1, 2012 and june 1, 2016 at king abdulaziz medical city in riyadh and king fahad general hospital in jeddah, were included. according to the rrt-pcr results, 135 patients were positive for mers-cov and 225 were negative. demographic characteristics, clinical presentations, and radiological and laboratory findings were collected for each subject. results: a risk prediction model to identify pneumonia patients at increased risk of mers-cov was developed. the model included male sex, contact with a sick patient or camel, diabetes, severe illness, low white blood cell (wbc) count, low alanine aminotransferase (alt), and high aspartate aminotransferase (ast). the model performed well in predicting mers-cov infection (area under the receiver operating characteristics curves (auc) 0.8162), on internal validation (auc 0.8037), and on a goodness-of-fit test (p = 0.592). the risk prediction model, which produced an optimal probability cut-off of 0.33, had a sensitivity of 0.716 and specificity of 0.783. conclusions: this study provides a simple, practical, and valid algorithm to identify pneumonia patients at increased risk of mers-cov infection. this risk prediction model could be useful for the early identification of patients at the highest risk of mers-cov infection. further validation of the prediction model on a large prospective cohort of representative patients with pneumonia is necessary. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in 2012. the diagnosis of this infection remains complex (al johani and hajeer, 2016; sung et al., 2016; ahmed, 2017a) and it has a high fatality rate (ahmed, 2017b, c; aleanizy et al., 2017; sherbini et al., 2017; kim et al., 2017) . the early detection and identification of individuals at high risk of a disease is the most effective strategy to improve patient clinical outcomes (ahmed, 2017a) and reduce the costs of testing, both physical and economic (ahmed et al., 2011 (ahmed et al., , 2013 . the real-time reverse transcription polymerase chain reaction (rrt-pcr) has been found to be valid and accurate for the evaluation of respiratory swabs, sputum, and other endotracheal aspirate material (corman et al., 2012a, b) . however, although rrt-pcr is the most accurate and sensitive test available at the authors' centers, absolute identification of mers-cov may require multiple clinical specimens from different sources and take days (corman et al., 2012a, b; anon, 2018a) . the saudi ministry of health (moh) has developed mers-cov visual triage guidelines to identify suspected cases (anon, 2018b) . the current guidelines include fever, respiratory symptoms, gastrointestinal symptoms, chronic diseases, and risk of exposure to mers-cov. in clinical practice, identifying high-risk individuals can be challenging, since most laboratory-confirmed mers-cov patients report common clinical risk indices to patients with other respiratory conditions. for instance, respiratory and gastrointestinal symptoms are common for both mers-cov and non-mers-cov patients (mohd et al., 2016) . thus, further exploration must take place to reduce the risk of mers-cov infection. a risk prediction model is urgently needed to stratify patients with suspected mers-cov. this may decrease the risk of virus transmission to others who are in close contact, for example healthcare workers, patients, and hospital visitors, by allowing the careful management of those who are potentially infected at an early stage of infection. developing a mers-cov prediction model may more efficiently aid physicians in identifying individuals at high risk and selecting the necessary test(s) to improve prevention and control measures. several methodological studies have shown that combining demographic characteristics with clinical, radiological, and laboratory information can improve risk assessment and diagnostic accuracy (ahmed et al., 2011 (ahmed et al., , 2013 sidransky, 2002; etzioni et al., 2003) . these previous studies used a linear combination to develop an algorithm that combines demographic characteristics, symptoms, and clinical, radiological, and laboratory findings to identify the highly suspected mers-cov cases. mers-cov was initially identified in a patient being treated for pneumonia in 2012 (zaki et al., 2012) , and since then, pneumonia and its symptoms have remained common characteristics in mers-cov patients (saad et al., 2014; choi et al., 2016) . the aim of this study was to develop and validate a reliable risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. a two-center, retrospective case-control study was conducted utilizing data from king abdulaziz medical city in riyadh (kamc-r) and king fahad general hospital in jeddah (kfgh-jed), saudi arabia. the data were obtained between september 1, 2012 and june 1, 2016. kfgh-jed experienced a mers outbreak between march and may 2014 (oboho et al., 2015) , and kamc-r experienced a large mers outbreak between june and august 2015 (al-dorzi et al., 2016) . both study centers applied the saudi moh case definitions (anon, 2018b) to identify suspected individuals in the population studied, and rrt-pcr was used as the gold standard test to diagnose mers-cov in multiple and different clinical specimens when necessary. mers-cov-related symptoms were common at both centers. the project received ethical approval from two independent ethics committees: the saudi moh (irb log number 16-230e) and king abdullah international medical research center (study number rc17/061), riyadh saudi arabia. during the study, the medical records of 829 subjects who were being assessed by rrt-pcr for suspected mers-cov were reviewed. the suspicion of mers-cov infection at both kamc-r and kfgh-jed was determined based on the saudi moh guidelines (anon, 2018b) . two groups were compared: patients who were positive and patients who were negative for mers-cov infection. in an effort to reduce heterogeneity between the study groups, only subjects with a lung infiltrate on chest x-ray and/or computed tomography (ct) scan of the chest were included in the analysis. thus, subjects who had no available results of a chest x-ray or ct scan of the chest were excluded. the initial screening for suspected mers-cov patients includes pneumonia (anon, 2018b) . most of the patients studied were evaluated for pneumonia immediately after presentation. the study excluded subjects who were less than 15 years of age (pediatrics/children), as defined in the saudi moh guidelines (anon, 2018b) , and excluded subjects who had upper respiratory tract infections (respiratory symptoms, positive or negative mers-cov rrt-pcr, and normal chest x-ray and ct scan of the chest). the final sample comprised a cohort of 360 subjects who had a lung infiltrate on chest x-ray and/or a ct scan of the chest, who were classified according to the results of mers-cov rrt-pcr. the case group consisted of 135 pneumonia patients who were positive for mers-cov infection, and the control group consisted of 225 pneumonia patients who were negative for mers-cov infection. cases were defined as patients with mers-cov pneumonia who had positive mers-cov rrt-pcr on nasopharyngeal aspirate and/ or bronchoalveolar lavage in addition to a lung infiltrate on chest xray and/or ct scan of the chest. controls were defined as patients with respiratory symptoms, a lung infiltrate on chest x-ray and/or ct scan of the chest, pneumonia, and negative mers-cov rrt-pcr result of nasopharyngeal aspirate and/or bronchoalveolar lavage. nineteen predictive variables were included: age, sex, fever (temperature !38 c), one composite respiratory symptom (the presence of cough, bloody cough, shortness of breath, or chest pain), one composite gastrointestinal symptoms (the presence of diarrhea, vomiting, or nausea), seven mers-cov potential risk factors (contact with sick patients or camels, severe illness (defined according to the patient's clinical status, 'yes/no', which is based on clinical judgment), diabetes, lung disease, liver disease, renal disease, and heart disease), and seven laboratory measurements (white blood cell (wbc) count (â10 9 /l), platelets (â10 9 /l), creatinine (mmol/l), bilirubin (mmol/l), alanine aminotransferase (alt; u/l), aspartate aminotransferase (ast; u/l), and albumin (g/ l)). the reference ranges for the laboratory measures were as follows: wbc count, 4-11 â10 9 /l; platelets, 150-400 â 10 9 /l; creatinine, 50-98 mmol/l; bilirubin, 3.4-20.5 mmol/l; alt, 5-55 u/l; ast, 5-34 u/l; albumin, 35-55 g/l. no serological tests were available at the centers for these patients. stata statistical software release 15, 2017 (statacorp. llc, college station, tx, usa) was used for the data analysis. the sample characteristics were recorded as the frequency and percentage, or as the mean ae standard deviation (sd). laboratory measurements were summarized as the median and 25th-75th percentiles. a subgroup analysis (chi-square test, independent samples t-test, or mann-whitney u-test) was used to identify unadjusted associations between demographic, clinical, and laboratory measurements according to mers-cov status. the performance of each bivariate predictor was further assessed by unbiased estimate, the area under the curve (auc), and its 95% confidence interval (ci). stepwise binary logistic regression was employed to develop a mers-cov risk prediction model and identify factors that could be used to estimate the probabilities of mers-cov infection. the goodness-of-fit of the final model was tested by hosmer-lemeshow procedure: a large p-value indicates that a model has a good fit. the discrimination ability between high and minimal risk of mers-cov infection of the final model was assessed by the auc and its 95% ci. a receiver operating characteristics (roc) curve was generated for the risk prediction model. two hundred random samples were drawn with replacements from the original study sample (n = 360). the model internal validity was assessed in these 200 samples by the auc and its 95% ci. optimal cut-off values of the probabilities for each model were determined using a generalized youden index (youden, 1950) . at these thresholds, it was possible to achieve the best balance between specificity and sensitivity. a total of 360 pneumonia patients were included in the analysis: 37.5% were confirmed mers-cov-positive and 62.5% were confirmed mers-cov-negative. the mean age at presentation was 55.9 years, with a standard deviation of 20.2 years; age ranged between 16 and 109 years. of the total sample, 6.9% had been in contact with a sick patient or camel, 60% had fever, 84.2% had at least one respiratory symptom, and 18.6% had at least one gastrointestinal symptom. the two groups were similar in the distribution of age (p = 0.220) and sex (p = 0.079). the characteristics of the sample can be found in table 1 . subgroup analyses are presented in tables 1 and 2. the chisquare test or the independent samples t-test revealed that sex (p = 0.079) and age (p = 0.220) were similar in the two groups. the risk of mers-cov infection was similar in patients with and without fever (p = 0.267), respiratory symptoms (p = 0.080), or gastrointestinal symptoms (p = 0.382). severe illness (p = 0.001), contact with a sick patient or camel (p = 0.001), diabetes (p = 0.001), heart disease (p = 0.007), and renal disease (p = 0.025) were significantly associated with an increased risk of mers-cov infection. the independent samples mann-whitney u-test revealed that the wbc count (p = 0.001) and platelet count (p = 0.006) were significantly lower in patients who were positive for mers-cov than in those who were negative for mers-cov infection. in contrast, creatinine (p = 0.001), bilirubin (p = 0.001), ast (p = 0.001), and albumin (p = 0.004) were significantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. alt (p = 0.352) was insignificantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. according to the individual roc curve analysis (table 3) , severe illness, diabetes, wbc count, creatinine, bilirubin, albumin, and ast were the most important predictors of mers-cov infection. a risk prediction model was developed using stepwise binary logistic regression (p 0.05). the model retained seven independent variables that were associated with increased odds of mers-cov (table 4 ). male sex (adjusted odds ratio (aor) 1.883, p = 0.043), contact with a sick patient or camel (aor 21.915, p = 0.001), diabetes (aor 2.703, p = 0.002), severe illness (aor 6.312, p = 0.001), low wbc count (aor 0.897, p = 0.001), high ast (aor 1.007, p = 0.007), and low alt (aor 0.995, p = 0.024) were found to have a significant impact on the prediction of mers-cov. the hosmer-lemeshow test indicated that this model fitted the data well (p = 0.592). this model showed substantial discrimination, with an auc of 0.8162 and a 95% ci of 0.7651-0.8674 ( figure 1 ). the prediction model was validated using the bootstrap procedure. a total of 200 random samples were drawn with replacements from the original sample, and the model showed a substantial ability to assess mers-cov infection in this study population (auc 0.804, 95% ci 0.7838-0.8235). the findings in table 4 were used to create a risk-probability model. the risk prediction for the model can be expressed by the following equation: predicted probability = [1 + exp(1.409-(0.633 â male) à (3.087 â sick patient or camel contact) à (0.995 â diabetes) à (1.842 â severe illness) + (0.109 â wbc count) à (0.007 â ast) + (0.005 â alt))] à1 . a calculator was developed to calculate the potential risk of mers-cov infection in pneumonia patients. we determined the optimal cut-off or threshold values of the probabilities to mark the differences between the high-risk and low-risk groups. when an equal weight was given for sensitivity and specificity (m = 1), the optimal cut-off value (probability !0.33) produced sensitivity and specificity of 0.716 and 0.783, respectively. when more weight was given for sensitivity than specificity (m = 0.50), the optimal cut-off value (probability !0.20) produced sensitivity and specificity of 0.902 and 0.550, respectively. when more weight was given for specificity than sensitivity (m = 1.50), the optimal cut-off value (probability !0.39) produced sensitivity and specificity of 0.647 and 0.831, respectively. based on data from the two largest hospitals in saudi arabia, a risk prediction model was developed for mers-cov infection in pneumonia patients. this model was generated from a retrospective study and should be assessed prospectively for external validation. seven variables were identified as having a great impact on the mers-cov risk assessment prediction. the risk prediction model highlights the strong potential impact of male sex, contact with a sick patient or camel, severe illness, diabetes, low wbc count, high ast, and low alt on mers-cov infection. these few important parameters could be part of routine medical examinations to be performed (for the purpose of identifying highly suspected individuals) in daily clinical practice in order to make a prompt and timely clinical decision. according to the model, high ast was associated with an increased risk of being infected with mers-cov. this finding is similar to that of mohd et al., who noted high ast levels in mers-cov patients (mohd et al., 2016) . unlike their findings, it was noted in the present study that the impact of alt became significantly negative after controlling for several confounders. however, this type of association should be evaluated further in a prospective study in the presence of other unmeasured confounders. although sex was found to have no impact on mers-cov infection in the unadjusted analysis, the multivariate analysis revealed that the risk of mers-cov infection was 88.3% times higher in males than in females. this may be because other factors are playing a role in the development of mers-cov in males, such as camel contact, since males are more likely than females to have contact with camels. in agreement with the recent saudi moh mers-cov visual triage guidelines for the identification of suspected cases (anon, 2018b) , it was found that the odds of being infected with mers-cov were higher in patients with diabetes as compared to those with no diabetes. this also supports the findings of previous studies (badawi and ryoo, 2016; assiri et al., 2013; al-tawfiq et al., 2014; alraddadi et al., 2016) in which researchers systematically recognized that diabetes is a risk factor for mers-cov infection. these findings indicate that more attention should be given to assessing the risk of mers-cov infection in diabetic patients and whether the risk depends on a specific diabetes type or condition in these patients. as asserted in the saudi moh mers-cov visual triage guidelines and many other studies (muhairi et al., 2016; younan et al., 2016; reeves et al., 2015; sabir et al., 2016; azhar et al., 2014a, b) , contact with a sick patient or camel was identified as an independent predictor of mers-cov infection, according to the risk prediction model. it must be noted that the finding in the present study could have been limited by combining camel contact and sick patient contact due to the small sample size of each category. this study shows the importance of incorporating various types of information to improve the performance of the risk prediction. according to the linear combination model, it was found that several of the parameters highlighted in the saudi moh mers-cov visual triage guidelines were not able to distinguish between 'highrisk' and 'low-risk' groups, nor did they help in predicting mers-cov infection. for instance, fever, respiratory symptoms, gastrointestinal symptoms, heart disease, and renal disease were noted to have an insignificant impact on mers-cov infection. however, in agreement with the saudi moh mers-cov guidelines and two other reports (mohd et al., 2016; arabi et al., 2017) , the odds of being infected with mers-cov were associated with a significant risk reduction of 10.3% for each unit increase in wbc count. these results suggest that demographic, clinical, radiological, and laboratory data should be used in routine practice to identify suspected mers-cov cases, as such data could serve as the first line of prevention strategies. it was found that the accuracy of prediction (figure 1 ) was further improved when combining various medical and patient variables as opposed to relying on a single factor (table 3 ). this has been proven theoretically and in application (ahmed et al., 2013 (ahmed et al., , 2011 (ahmed et al., , 2015 etzioni et al., 2003; shen, 2008; pepe and thompson, 2000; su and liu, 1993; thompson, 2003) , where a linear combination has been used to maximize the diagnostic accuracy of a disease of interest. the strength of this study lies in the fact that a simple and applicable predictive model was developed that incorporates demographic, clinical, radiological, and laboratory data, where these were functionally associated and contributed greatly to stratifying and distinguishing between a high and a minimal risk of mers-cov infection. this simple evaluation of suspected mers-cov cases appears promising and could be implemented easily in routine clinical practice. this model could be used as a risk stratification method or a triage tool to help physicians in making an informed decision and planning the next step when deciding whether an rrt-pcr or further investigation is necessary. it was possible to derive a risk probability algorithm (range 0-1), a generalized youden index (choi et al., 2016) was used to determine an optimal cut-off to stratify the risk, and a risk probability of !0.41 was identified as being the optimal cut-off, with a sensitivity of 0.688 and specificity of 0.789. several limitations to the proposed risk prediction model were identified. the study findings were based on a retrospective design; therefore this prediction model should be interpreted with caution. limited information was available on patient variables, clinical variables, and transmission routes. for example, information on primary cases and secondary cases may be supplemented by the results of clinical, radiological, and laboratory data. in this study, 'contact with a sick patient' and 'contact with a sick camel' were combined into one variable due to the small number in each category. severe illness was based on a subjective judgment. an additional potential limitation was that the duration of symptoms was not available for these patients. this study investigated a very specific population (pneumonia) at only two centers, which could compromise the applicability and generalizability of the risk prediction model. moreover, the prediction model may not be generalizable to patients who do not fulfill the moh guidelines. further validation of the prediction model on an external sample and prospective cohort of representative patients with pneumonia is necessary, specifically in relevant settings: emergency, outpatient, inpatient, and community. despite these limitations, the model developed shows promise for the identification of suspected mers-cov cases in clinical practice. this model could be applicable in various healthcare settingsinpatient, outpatient, and emergency departmentsbecause no extensive laboratory testing is required and samples may be available within short turnaround times. this may allow rapid evaluation and improve clinical decision-making. in conclusion, this study provides a simple, practical, and valid algorithm to identify individuals at increased risk of mers-cov infection among patients who have developed pneumonia. this risk model is not only useful for risk stratification and decisionmaking in clinical practice, but it could also be useful in preventing and managing the possible spread of mers-cov. the usefulness of this newly developed tool most be validated in an external prospective study. the project received ethical approval from two independent ethics committees: the saudi ministry of health (irb log number 16-230e) and king abdullah international medical research center (study number rc17/061), riyadh saudi arabia. none. none declared. accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach believe the extreme (be) strategy at the optimal point: what strategy will it become diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study the predictors of 3-and 30-day mortality in 660 mers-cov patients mers-cov diagnosis: an update the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study risk factors for primary middle east respiratory syndrome coronavirus illness in humans laboratory testing for middle east respiratory syndrome coronavirus (mers-cov) case definition and surveillance guidance -updated critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient evidence for camel-to-human transmission of mers coronavirus prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction combining biomarkers to detect disease with application to prostate cancer middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate mers-cov outbreak in jeddah-a link to health care facilities combining diagnostic test results to increase accuracy mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia on the principles of believe the positive and believe the negative for diagnosis using two continuous tests middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data emerging molecular markers of cancer linear combinations of multiple diagnostic markers comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription pcr assessing the diagnostic accuracy of a sequence of tests index for rating diagnostic tests mers and the dromedary camel trade between africa and the middle east isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors acknowledge the saudi ministry of health and king abdullah international medical research center for approving this research project. the authors would like to thank the leaders of king abdulaziz medical city in riyadh and king fahad general hospital in jeddah for their support and understanding. supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ijid.2018.03.005. key: cord-272513-umuiovrd authors: bindayna, khalid mubarak; crinion, shane title: variant analysis of sars-cov-2 genomes in the middle east date: 2020-10-09 journal: biorxiv doi: 10.1101/2020.10.09.332692 sha: doc_id: 272513 cord_uid: umuiovrd background coronavirus (covid-19) was introduced into society in late 2019 and has now reached over 26 million cases and 850,000 deaths. the middle east has a death toll of ∼50,000 and over 20,000 of these are in iran, which has over 350,000 confirmed cases. we expect that iranian cases caused outbreaks in the neighbouring countries and that variant mapping and phylogenetic analysis can be used to prove this. we also aim to analyse the variants of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) to characterise the common genome variants and provide useful data in the global effort to prevent further spread of covid-19. methods the approach uses bioinformatics approaches including multiple sequence alignment, variant calling and annotation and phylogenetic analysis to identify the genomic variants found in the region. the approach uses 122 samples from the 13 countries of the middle east sourced from the global initiative on sharing all influenza data (gisaid). findings we identified 2200 distinct genome variants including 129 downstream gene variants, 298 frame shift variants, 789 missense variants, 1 start lost, 13 start gained, 1 stop lost, 249 synonymous variants and 720 upstream gene variants. the most common, high impact variants were 10818deltinsg, 2772delcinsc, 14159delcinsc and 2789delainsa. variant alignment and phylogenetic tree generation indicates that samples from iran likely introduced covid-19 to the rest of the middle east. interpretation the phylogenetic and variant analysis provides unique insight into mutation types in genomes. initial introduction of covid-19 was most likely due to iranian transmission. some countries show evidence of novel mutations and unique strains. increased time in small populations is likely to contribute to more unique genomes. this study provides more in depth analysis of the variants affecting in the region than any other study. funding none on january 9 h 2020, the china centre for disease control reported that 15 of 59 suspected cases of pneumonia were due to a novel human coronavirus (cov), now known as severe acute respiratory syndrome cov 2 (sars-cov-2) 1 . the genome for this novel virus was then made publicly available on the global initiative on sharing all influenza data (gisaid) the next day. sars-cov-2 is an easily spreadable virus which would evolve into a global pandemic of at least 26 million cases and 850,000 deaths 2 . one of the first countries to experience a significant outbreak was iran. the country reported its first confirmed case on 19 th february 2020 from a merchant in qom who travelled from china 3. . many of the first countries with infections in the middle east were linked to travellers from iran including lebanon, kuwait, bahrain, iraq, oman and uae. covid-19 continued to spread to the remaining middle eastern countries with a death toll of over 50,000 people according to health authorities. this number is expected to be an underestimation due to countries effected by war including libya, syria and yemen. needless to say, there have been devastating effects to the region and the real effects are expected to be unreported 4 . researchers are racing to develop a vaccine that can provide viral immunity and avoid additional deaths. sar-cov-2 is transmitted using the spike protein which binds to human angiotensin-converting enzyme 2 (ace2) receptor; the virus is easily transmittable due to mutations in the receptor-binding (s1) and fusion (s2) domain of the strain 5 . transmission could be made even easier if more mutations accumulate. although mutations are rare, they can create new strains and it is not guaranteed that the current leading vaccine trials will be effective as sars-cov-2 continues to mutate 6 . by categorizing variants, we can identify any new strains and how the mutations are likely to affect spread. as the middle east is often under reported, it is important to characterise the variants of strains that are commonly present. analysis of the common variants in the middle east is essential to develop a vaccine that treats the strains in the region. this analysis helps understand the viral genome landscape and identify clades of the region. • our hypothesis is that variants found in sars-cov-2 genomes from middle eastern samples will indicate delivery from iran. we will use bioinformatics tools and publicly available samples to explore the composition of strains within each country. we expect that many strains will show evidence of iranian origin. • the aim is to explore the structure of middle eastern genome strains using multiple sequence alignment, tree generation and variant prediction (and others). if we explore the structure and common variants of sars-cov-2 strains in these populations, we expect to learn more about how the virus spread. sample source: we obtained the publicly available data from the global initiative on sharing all influenza data (gisaid) 7 . samples were also filtered to high quality when possible. 10 samples was selected as the optimum number to cover all possible countries and remain within alignment file limit of size 4 mb (maximum size for clustal omega tool). in countries with 10 samples, to prevent sample sourcing from same outbreak, the 5 earliest and 5 most recent samples were taken. all samples were downloaded from gisaid and then concatenated into a single multi-sample file and saved in fasta format. multiple sequence alignment: using the collected samples, multiple sequence alignment (msa) was performed using clustal omega (https://www.ebi.ac.uk/tools/ msa/clustalo/) 8 . the clustal omega online tool was used to perform the alignment (found at: https://www.ebi.ac.uk/tools/msa/clustalo/). the online tools allows up to 4000 sequences or a maximum file size of 4 mb, therefore the maximum number of samples was used. the concatenated file of samples was uploaded to the online tool. for step 2, the output parameters selected were pearson/fasta. all other options were kept at the default option. the output file generated is an alignment file; the file consists of all sequences with gaps denoted by '-'. the output file format is also a fasta file. variant identification: variant calling was performed using the alignment fasta file and the snp extraction tool snp-sites 9 (https://github.com/sanger-pathogens/snp-sites). these tools identify the snp sites by taking a multi-sample fasta file as input. the program then restructures the data as a variant call format (vcf) file. the vcf file provides a clear mapping of snps from the aligned sequences -this allows us to easily identify the snp location and the genotype for each sample at a given locus. in the outputted vcf file, the rows correspond with each unique variant and the column provides the genotype at the given site. we used snp-eff 10 to perform the variant annotation information such as the variant definition and the overlapping gene (found at: (https://pcingola.github.io/snpeff/snpeff.html). snpeff also predicts the effect of the variants. snpeff is integrated into the galaxy web-based tool for bioinformatics analysis (found at: usegalaxy.org) 11 . we utilised the galaxy platform and uploaded our vcf file to galaxy using their online upload tool. to annotate variants, you must first build a database from the reference genome. this is performed using the "snpeff build" tool on galaxy. to create the snpeff database, we downloaded data from ncbi for wuhan reference nc_045512.2 and uploaded to galaxy. to build the database, we directed to ncbi and searched for nc_045512. we then downloaded the corresponding gff file, which contains the annotations, and the fasta file, which contains the entire genome. we then selected the build database option. once the database was built, we selected the "snpeff eff" tool to annotate variants. galaxy populates the fields for vcf with the uploaded file from the previous step. the output format is selected as vcf and csv report was also selected for additional useful information for downstream analysis. for genome source parameter, the option "custom" was selected to use the newly created database. other default parameters were used including 5000 bases for upstream / downstream length and 2 bases as set size for splice sites (donors and acceptor) in bases. all filter output and additional annotation options were deselected and analysis was ran. once the analysis was executed, the annotation data is outputted as an annotated vcf and a html report file. data visualisation: once the annotated vcf was generated, the vcf was imported to r for extraction of the variant annotation information. the annotated data was imported, manipulated and plotted using r v3.6.2 12 . the dplyr v0.8.4 package was used to summarise and align the data 13 . the ggplot2 package was used to align the identified variants and visualise the types of mutations that re-occur 14 . the x-axis in the plots indicates the variant position along the sars-cov-2 genome; the left y-axis indicates the sample name and the right y-axis represents the country of origin for each sample. this plot is used to compare the genome in different populations. the data was then ordered by date of first reported case, meaning that wuhan is followed by united arab emirates and the final country is cyprus. phylogenetic analysis: phylogenetic analysis was performed using beast (bayesian evolutionary analysis sample trees) v1.10.4, to perform bayesian analysis of molecular sequences using monte carlo markov chains (mcmc) 15 . the analysis followed the approach recommended to reconstruct the evolutionary dynamics of an epidemic. the aim of this is to obtain an estimate of the origin of the epidemic in the region and understand how it spread through the middle east. to undertake the analysis, we opened beauti, the graphical application used to analyse the control file. although it requests a nexus file, the fasta file can also be used. the data was uploaded using the import data option and appeared under the partitions section. beauti confirmed that 30851 sites are present in the uploaded data. the default options are selected for site model and clock model. next, we specified the individual virus dates by selecting the "tips" panel and selecting the "use tip dates" option. a tab delimited file was uploaded which specified the upload date. this information was extracted from the names as they were downloaded from gisaid. next, we set the substitution model by selecting the "sites" tab and selected the default options of hky model, the default estimated base frequencies and select gamma as site heterogeneity model. next, the molecular clock was selected under the "clock" tab as a strict clock since we know that the frequency of mutation is low. the tree options are elected under the "tree prior" tab as "random starting tree" for the tree model and "coalescent: exponential growth", a model that assumes a finite but constant population size and predicts that all alleles will be removed from the population individually. this provides additional predictions on the reproductive rate. in the "priors" tab, select the scale as 100 for prior distribution which models the expected growth for a pandemic. the operators require no changes from the default. the mcmc option for chain length is set as 100,000 and sampling frequency to 100. tree visualisation: finally, we summarised the tree using the treeannotator tool, an additional package as part of beast. we first select the file generated using beast and outputted the tree file. then the output nexus file was imported to figtree program to display. once we opened figtree to display, we re-ordered the order by increasing value and then switched on branch labels. we switched on node bars and selected the 95% highest posterior density (hpd) credible intervals for the node heights. we plotted a time scale by turning on the scale axis and then setting the time scale section for offset as 2020.7, the latest date of collection for our samples. sequence alignment and variant calling were completed successfully. once these were complete, variation annotation was performed. we identified 2200 distinct genome variants which are recorded in table 1 . the most common, high impact variants were 10818deltinsg, 2772delcinsc, 14159delcinsc and 2789delainsa. the frequency of each unique variant type can be found in table 2 , which outlines the locus of all snps with over 50 instances. frame shift variants 298 start lost 1 stop lost 1 upstream gene variant 720 these results were then used to generate the dotplot of variant alignment (figure 1 ). the dotplot successfully indicated a pattern in variants that could not be easily identified from the alignment or annotation files. the alignment includes samples in facets based on their country. the alignment shows a pattern in variants that occur between each country. for example, this is prominent in qatar, jordan and oman where the pattern makes the country distinctive from the variants plotted for other countries. in addition, the phylogenetic tree generated branching indicative of an iranian origin ( figure 2 ) the aim of this study was to identify whether covid-19 was introduced to the middle east from iran and also to explore the genomic composition in the region. our study performs sequence alignment to compare all sequences against the reference genome. once this is complete, the annotated variants were extracted to generate a plot mapping variants, grouping samples by country. the plot as seen in figure 1 , shows clear distinctive patterns within countries that are not obvious from the generated alignment and annotation files. this may indicate a diverse, new strain is circulating in the country. cyprus has little diversity in the variant mapping which is surprising given its late date for first reported cases. another interesting point is that time-varied samples were taken for countries with 10 samples. we see not indication that there are distinct groups within countries. this further indicates the the mutation frequency is low. it also indicates that there is more variation in the genomic composition in samples from different countries than differences found in samples from different collection times. smaller populations can cause greater accumulation of variants through genetic drift. this may occur given local lockdowns and travel restrictions that have been enforced worldwide. it is possible that these genomic strains with new mutations may create a situation where the countries develop a deathly strain that is not prominent in other parts of the world. this could result in a situation where a country is disproportionately affected by accumulating deaths or an inefficient vaccine. phylogenetic trees help in understanding the evolutionary relationships between groups. in the present context, we use them to identify the earliest strains and to track the spread of covid-19 across the middle east. the tree shows that uae samples are distinguished and form one clade. this correlates with their early intervention and lockdown and subsequently appears to have resulted in a unique genome. samples from qatar also form the majority of 1 clade, with many of the wuhan samples, indicating that they are similar to the wuhan samples and show little distinction. egypt also becomes a distinct branch earlier than most samples. these examples are indicative of the global response -the lockdown of each country and prevention of spreading has resulted in sars-cov-2 strains of great similarity within each country. if lockdowns were not enforced, it is likely that these clades would be less distinguisable as mutations are spread between countries. as we expected, 2 of the highest branches points attach to iranian samples, further implementing iran in the initial spread across the middle east. the phylogenetic tree therefore indicates what we suggest in our hypothesis -most samples originate from the iranian sample. this is not surprising given the vast number of cases and early crisis state of the country. however, it is useful to see that the variant analysis shows what we suspect at the genome level. a related study also came to this conclusion by using contact tracing from cases related to religious events in the city of qom, iran 16 . risk assessment: outbreak of acute respiratory syndrome associated with a novel coronavirus covid-19 situation reports mapping the incidence of the covid-19 hotspot in iran -implications for travellers challenges to testing covid-19 in conflict zones: yemen as an example phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop on the origin and continuing evolution of sars-cov-2 data, disease and diplomacy: gisaid's innovative contribution to global health fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega snp-sites: rapid efficient extraction of snps from multi-fasta alignments a program for annotating and predicting the effects of single nucleotide polymorphisms, snpeff: snps in the genome of drosophila melanogaster strain w1118 the galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update r: a language and environment for statistical computing. r foundation for statistical computing dplyr: a grammar of data manipulation ggplot2: elegant graphics for data analysis bayesian phylogenetic and phylodynamic data integration using beast 1.10 virus evolution 4 is visiting qom spread covid-19 epidemic in the middle east? the authors are grateful for the timely sequencing and release of genomes to make this study possible and to dr. anusha c p for her comments. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. iran epi_isl_424349 30 iran epi_isl_445088 31 iran epi_isl_507007 32 iran epi_isl_514753 33 iran epi_isl_437512 34 iran epi_isl_442044 35 iran epi_isl_442523 36 israel epi_isl_435291 37 israel epi_isl_514303 38 israel epi_isl_514305 39 israel epi_isl_514302 40 israel epi_isl_435286 41 israel epi_isl_435289 42 israel epi_isl_419211 43 israel epi_isl_435284 44 israel epi_isl_514306 45 israel epi_isl_514301 46 jordan epi_isl_430012 47 jordan epi_isl_430002 saudiarabia epi_isl_512924 93 saudiarabia epi_isl_512926 94 saudiarabia epi_isl_489996 95 saudiarabia epi_isl_489998 96 saudiarabia epi_isl_490000 97 saudiarabia epi_isl_489999 98 saudiarabia epi_isl_489997 99 saudiarabia epi_isl_512927 100 saudiarabia epi_isl_512922 101 saudiarabia epi_isl_512923 102 turkey epi_isl_495421 103 turkey epi_isl_495445 104 turkey epi_isl_495433 105 turkey epi_isl_429868 106 turkey epi_isl_429867 107 turkey epi_isl_429866 108 turkey epi_isl_428712 109 turkey epi_isl_495436 110 turkey epi_isl_495429 111 turkey epi_isl_424366 112 unitedarabemirates epi_isl_469277 113 unitedarabemirates epi_isl_469279 114 unitedarabemirates epi_isl_435126 115 unitedarabemirates epi_isl_435121 116 unitedarabemirates epi_isl_435131 117 unitedarabemirates epi_isl_463740 118 unitedarabemirates epi_isl_469281 119 unitedarabemirates epi_isl_469280 120 unitedarabemirates epi_isl_435137 121 unitedarabemirates epi_isl_435134 122 wuhan epi_isl_402123 123 wuhan epi_isl_454949 124 wuhan epi_isl_454948 125 wuhan epi_isl_406798 126 wuhan epi_isl_454951 127 wuhan epi_isl_403931 table 3 : catalogue of sample accession id by country. key: cord-309621-6jj19xpr authors: yu, pin; xu, yanfeng; deng, wei; bao, linlin; huang, lan; xu, yuhuan; yao, yanfeng; qin, chuan title: comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus date: 2017-02-24 journal: plos one doi: 10.1371/journal.pone.0172093 sha: doc_id: 309621 cord_uid: 6jj19xpr middle east respiratory syndrome (mers), which is caused by a newly discovered coronavirus (cov), has recently emerged. it causes severe viral pneumonia and is associated with a high fatality rate. however, the pathogenesis, comparative pathology and inflammatory cell response of rhesus macaques and common marmosets experimentally infected with mers-cov are unknown. we describe the histopathological, immunohistochemical, and ultrastructural findings from rhesus macaque and common marmoset animal models of mers-cov infection. the main histopathological findings in the lungs of rhesus macaques and common marmosets were varying degrees of pulmonary lesions, including pneumonia, pulmonary oedema, haemorrhage, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. the characteristic inflammatory cells in the lungs of rhesus macaques and common marmosets were eosinophils and neutrophils, respectively. based on these observations, the lungs of rhesus macaques and common marmosets appeared to develop chronic and acute pneumonia, respectively. mers-cov antigens and viral rna were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, and ultrastructural observations showed that viral protein was found in type ii pneumocytes and inflammatory cells in both species. correspondingly, the entry receptor ddp4 was found in type i and ii pneumocytes, bronchial epithelial cells, and alveolar macrophages. the rhesus macaque and common marmoset animal models of mers-cov can be used as a tool to mimic the oncome of mers-cov infections in humans. these models can help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the middle east respiratory syndrome coronavirus (mers-cov) was identified in 2012 in a cell culture taken from a patient who died of pneumonia in saudi arabia [1] . more men than women have become infected with this virus, and the median age of those affected is 47 years old (range: 9 months-94 years), with most of the fatalities occurring in patients over 60 years old [2, 3] . the respiratory symptoms of this infection are primarily related to severe lower respiratory tract complications (e.g., dyspnoea and coughing associated with a fever) that may become fatal, while there is generally little involvement of the upper respiratory tract. a large proportion of severely ill patients require mechanical ventilation [4, 5] . complications that have been described in fatal cases include hyperkalaemia with associated ventricular tachycardia, disseminated intravascular coagulation leading to cardiac arrest, pericarditis and multiorgan failure [6] . mers-cov seems to be widely present in dromedary camels in the middle east and in some parts of africa [7, 8] . zoonotic transmission is likely to have originated from this species and is expected to continue indefinitely in these regions. the entry receptor for mers-cov, dipeptidyl peptidase 4 (ddp4), also named cd26, shows a high similarity in both humans and dromedary camels. most mers patients acquire the infection in the middle east, which subsequently leads to limited human-to-human transmission in local groups and healthcare workers and eventually to travel-related cases outside the region, all of which can result in a mild to severe or even fatal respiratory disease [2] . finding a suitable animal model is a major challenge for understanding the pathogenesis of mers-cov infection, evaluating the safety and efficacy of mers-cov vaccine candidates and developing therapeutic interventions. experimental infections with mers-cov in rhesus macaques (macaca mulatta) [9] , common marmosets (callithrix jacchus) [10] , rabbits (oryctolagus cuniculus) [11] and mice (mus musculus) [12, 13] have been reported in studies on the pathological changes that occur as a result of this viral infection. however, little is known about the pathological changes in the lungs of humans infected with mers-cov, which makes it difficult to interpret data from experimental mers-cov animal models. overall, based on the known clinical aspects of mers-cov infection in humans, useful experimental animal models of mers-cov infection should exhibit a life-threatening lower respiratory tract disease. although there have been several studies in animal models on the pathogenic mechanisms of mers-cov infection, little is known about the comparative pathology and inflammatory cell response in rhesus macaques or common marmosets infected with this virus. therefore, it is vital to study comparative pathology on the association of the mers-cov antigen with its receptor, ddp4, or the histopathological changes in nonhuman primate (nhp) models of mers-cov infection. here, we comprehensively describe the histopathological features of the disease and the distribution of the mers-cov antigen and ddp4 in rhesus macaque and common marmoset models. our findings may contribute to a better understanding of the pathogenic process of mers-cov infection and help in evaluating the suitability and efficacy of the animal models used in the development of effective medications and prophylactic treatments for this disease. this research on the mers-cov virus was discussed among the staff members of the department of pathogen biology at the institute of laboratory animal science (ilas) of the chinese academy of medical sciences and peking union medical college (pumc). the experiments and protocols for this nhp models of mers-cov infection were discussed explicitly and extensively among the staff members of the department of pathogen biology. these discussions were followed by consultations with biosafety officers and facility managers at the ilas of pumc, as well as with numerous specialists in the fields of sars-cov and general infectious disease research throughout china. all research procedures were approved by the ilas institutional animal care and use committee and the laboratory safety committee (lsc). the committee recommended that the number of animals be reduced to comply with the 3r (reduction, replacement, refinement) principles. therefore, our experiment was designed to include three rhesus macaques and three common marmosets to test their effectiveness as animal models of mers-cov infection. two rhesus macaques and two common marmosets were infected with the virus and one individual of each species was left uninfected to serve as a control. the animals were planned to be euthaniazed when they were suffering from fatal respiratory symptom, impending death or 20% of body weight loss, which included fatal dyspnea and infectious shock. the approved registration number for this study is ilas-pc-2013-004. all experiments were conducted within an animal biosafety level 3 (absl-3) facility, which was constructed and accredited based on national standard gb19489 at the ilas of pumc, beijing, china. rhesus macaques and common marmosets were housed in accordance with chinese national standards, which are consistent with the standard set forth in the 8th edition of the nrc guide for the care and use of laboratory animals. because of the infectious nature of this study, nhps were housed individually instead of the generally recommended group or social housing. stainless steel cages measuring 0.5-0.75 m 2 and 0.5-0.6 m depending on the weight of the individual animal, consisted of wire flooring and resting boards or perches. rooms have natural lighting and the photoperiod is supplemented during the winter months with an artificial lighting source to provide a 12: 12 light cycle. temperature and humidity in animal holding rooms are maintained in accordance with recommendations in the chinese national standards for animal care. drinking potable water is obtained from the city of beijing and delivered to the animals via automated watering system (aws). the aws is checked daily to ensure proper operation i.e., water pressure, free flowing exits and absence of leakages. pans were cleaned daily and cages were washed every week by hand. all animals have individual cage id cards which contain the following basic information: study no., sex, weight, principal investigator's name and study protocol number. nhps were fed a measured amount of a commercially available nhp diet (beijing hfk bioscience co., ltd) offered twice daily. fresh fruit (apples, bananas and oranges) are supplemented on alternating days. additional environmental enrichment consists of toys, stainless steel mirrors and heavy-duty dog chew toys (nyla bones or similar), which are provided on a rotating basis. toys are left inside the cages when these are transported out of the room for washing and are sanitized at this time. damaged toys are removed from circulation. soft background music, plants, as well as pictures and photos hung on the animal room walls are provided for relaxation. opportunities for limited social interaction with compatible nhps are also provided at every other cage change when cages of compatible animals are placed in close proximity to each other while avoiding direct physical contact between animals. two rhesus macaques, 2-3 years old, were anesthetised with ketamine hydrochloride (30 mg/kg, i.m) prior to the procedures and intratracheally inoculated with 1 ml of hcov-emc (6.5 ×10 7 tcid 50 /1 ml) diluted in dmem. one mock-infected rhesus macaque was intratracheally inoculated with tissue culture media dmem for use as control. the rhesus macaques were observed twice daily, and clinical signs were recorded. the infected and mock-infected rhesus macaques were anesthetized with pentobarbital sodium (60 mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at 3 days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. two common marmosets, 2-3 years old, were anesthetised with ketamine hydrochloride (120 mg/kg, i.p) prior to the procedures and intratracheally inoculated with 1 ml of hco-v-emc (5 ×10 6 tcid 50 /0.5 ml) diluted in dmem. one mock-infected common marmoset was intratracheally inoculated with tissue culture media dmem for use as control. the common marmosets were observed twice daily, and clinical signs were recorded. the infected and mock-infected common marmosets were anesthetized with pentobarbital sodium (40 mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at 3 days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. none of the infected animals were euthanized or died without euthanasia prior to their sacrifice at 3 days post-infection. the fixed samples were dehydrated and dewaxed according to conventional procedures, and 4-μm sections were prepared with a microtome. some sections were stained with haematoxylin-eosin (he) using routine methods. two independent pathologists observed all slides and were blinded to the experimental design. lungs were fixed in glutaraldehyde and prepared for ultrastructural observations. transmission electron microscopy was performed essentially as previously described [14] . briefly, serial sections were dewaxed and rehydrated in graded ethanol, and a standard avidinbiotin immunoperoxidase technique was performed [15] . table 1 lists the primary antibodies used for ihc. optimal antibody dilutions were determined in experiments on positive control tissues. negative control sections were prepared using the same steps as described above, but the primary antibodies were derived from an irrelevant sera. sections were dewaxed and rehydrated in a graded ethanol series. ish was carried out using the enhanced sensitive ish detection kit i (boster, china) according to the manufacturer's instructions. endogenous peroxidase activity was quenched with 0.5% hydrogen peroxide in methanol at room temperature for 30 minutes. proteinase k digestion was performed at 37˚c for 20 min. then, pre-hybridization was performed at 37˚c for 3 hours. after removing excess pre-hybridization buffer, 2 μg/ml digoxin (dig)-modified oligo-nucleotide antisense probes (table 2) in the hybridization solution were applied to the sections, followed by incubation at 37˚c overnight. after washing the slides in 2× saline-sodium citrate (ssc), 0.5×ssc, and 0.2×ssc buffer, the sections were incubated in a blocking buffer at 37˚c for 30 min. the sections were then incubated with biotinylated mouse anti-dig at 37˚c for 60 min and with streptavidin biotin peroxidase and biotinylated peroxidase for an additional 20 min, with each incubation followed by three washes in phosphate-buffered saline (pbs). the sections were treated with 3, 3-diaminobenzidine for 2 min, counterstained in haematoxylin for 5 min, dehydrated, and mounted with neutral gum. sections for the negative controls were prepared using the same steps described above, but the antisense or sense probes were replaced with pbs at ph 7.4. rhesus macaques were observed twice daily for clinical signs. the rectal temperature of the infected rhesus macaques increased to 40.5˚c at 1-2 days post-infection, and thereafter turned to normal. the infected common marmosets showed manifest symtoms of viral infection, including severe respiratory symtoms, drastical water intake decrease and overt weight loss, and the maximum body weight loss were about 11%. none of the mock infected nhps showed abnormal clinical signs or died during the expriment. pathological findings in the rhesus macaque tissues he stained tissues from rhesus macaques experimentally infected with mers-cov demonstrate that mers-cov induces lesions that are primarily observed in the lungs, with varying degrees of inflammation, interstitial pneumonia (fig 1a) , pulmonary oedema (fig 1b) , haemorrhaging, degeneration and necrosis of pneumocytes and bronchial epithelial cells (fig 1c) , and the infiltration of inflammatory cells. focal interstitial pneumonia and pulmonary oedema were observed in different parts of the pulmonary lobes, as was mild haemorrhaging. the most prominent pathological effect observed in the lungs of rhesus macaques was diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control rhesus macaque (s1a fig) . pathological findings in common marmoset tissues a histopathological analysis detected numerous lesions in the lungs of the infected marmosets. exudative pathological changes were found, exhibiting haemorrhage, widespread pulmonary oedema and a large number of inflammatory cells. fibrinous exudates were observed in the oedematous alveolar cavities (fig 1d) . diffuse and focal neutrophil infiltration was found in the oedematous alveolar cavities (fig 1e) , bronchial lumen, and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells (fig 1f) . no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control common marmoset (s1b fig) . to investigate the infiltration of specific inflammatory cells, ihc was carried out to identify cd68+ macrophages, cd15+ neutrophils, cd57+ natural killer cells, cd20+ b lymphocytes, cd138+ plasma cells, and cd3+, cd4+, cd8+ t lymphocytes. in the lungs of both species, the diffuse infiltration of numerous macrophages (fig 2b and 2d ) was observed in the expanded alveolar septum and the oedematous alveolar cavities. however, in the lungs of rhesus macaques, a large number of diffusely and focally infiltrating eosinophils (fig 2a) were found in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. however, in the lungs of common marmosets, numerous neutrophils ( fig 2c) infiltrated into the oedematous alveolar cavities. in both of the nhp models, other types of inflammatory cells were rarely observed. using immunohistochemical techniques and an ish analysis, we confirmed that mers-cov protein and viral rna were distributed in the lungs of rhesus macaques and common marmosets and that they were primarily located in the pneumocytes and inflammatory cells. in the lungs of rhesus macaques, mers-cov antigens were extensively distributed in type i and ii pneumocytes, alveolar macrophages (fig 3a) , eosinophils and bronchial epithelial cells ( fig 3b) . from the microscopic characteristics, the cuboidal type ii pneumocytes are located on the alveolar cavities, and smaller than macrophages. viral rna was also distributed in pneumocytes and inflammatory cells in the lungs of rhesus macaques (fig 3c) . in the lungs of common marmosets, a moderate level of mers-cov-positive antigens were detected in pneumocytes, and antigens were found more extensively in alveolar macrophages (fig 3d) , especially in the inflammatory cells around the bronchus (fig 3e) . viral rna was massively distributed in pneumocytes and inflammatory cells in the lungs of common marmosets (fig 3f) . no mers--cov-positive antigens or viral rna was detected in the lungs of the control nhps (data not shown). pathological lesions and virus distribution in rhesus macaque and common marmoset animal models are summarized and shown in table 3 . to further determine the effects of mers-cov infection and replication in the lungs of common marmosets, ultrastructural observations were performed on lesions in infected lung samples and on mock-infected samples. virus particles were found in type ii pneumocytes ( fig 4a-4c ) and in inflammatory cells (fig 4d-4f) . under the electron microscope, the characteristic of type ii pneumocytes is lamellar bodies (s2 fig). no viral particles were observed in the lungs of mock-infected common marmosets (data not shown). to elucidate the relationship between mers-cov and its entry receptor, ddp4, we determined the expression pattern of ddp4 in the lungs of rhesus macaques and common marmosets using immunohistochemical techniques. we found that in the lungs of rhesus macaques, ddp4 was strongly expressed in type i and ii pneumocytes, bronchial epithelial cells (fig 5a) , and inflammatory cells, primarily alveolar macrophages (fig 5a) . similarly, in the lungs of common marmosets, ddp4 was widely expressed in type i and ii pneumocytes and alveolar macrophages (fig 5b) . however, ddp4 was only weakly expressed in the bronchial epithelial cells, mainly in basal and ciliated cells (fig 5b) . in the present study, we analysed the histopathological features of mers-cov infection in rhesus macaques and common marmosets. moreover, we compared the distribution of mers-cov antigens, viral rna and ddp4 expression in the infected lungs of these species. we found that the lungs of both species exhibited varying degrees of lesions, including pneumonia, pulmonary oedema, haemorrhaging, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. comparing the different trends in the two nhp models, it can be seen that varying degrees of inflammation, especially interstitial pneumonia, were found in the lungs of rhesus macaques, indicating mild disease and trend of chronic pneumonia; however, in the lungs of common marmosets, exudative pathological changes were found, exhibiting pulmonary oedema, inflammatory cell infiltration and fibrinous exudates, suggesting acute pneumonia. similar to our results, previous study have also reported that rhesus macaques developed mild disease, and common marmoset exhibited potentially lethal disease [16] . however, in our study we found that the prominent inflammatory cells in the two nhp models were different, which may be the causality of process in mers-cov infection. in our study, the diffuse infiltration of numerous macrophages was observed in the expanded alveolar septa and oedematous alveolar cavities of both species. however, the most prominent pathological effect observed in the lungs of rhesus macaques was a diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. in contrast, in the lungs of common marmosets, diffuse and focal neutrophil infiltration occurred in the oedematous alveolar cavities, bronchial lumen and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells. these differences in inflammatory cell infiltration suggest that inflammatory cells may function in the development of mers-cov infection. additionally, it is worth noting that eosinophils and neutrophils play important roles in rhesus macaques and common marmosets, respectively, in the development of pulmonary lesions and the pathogenesis of mers-cov infection. in the lungs of common marmoset, pulmonary oedema exhibited much more severe than that in the lungs of rhesus macaques, which may be due to the difference of inflammatory cells in the lungs of nhp models. similar to our results, previous studies have also reported that common marmosets infected with mers-cov exhibit acute bronchointerstitial pneumonia centred at doi:10.1371/journal.pone.0172093.g003 table 3 . pathological lesions and virus distribution in rhesus macaque and common marmoset animal models. pathology of mers-cov infection the terminal bronchioles, with an influx of inflammatory cells, a thickening of alveolar septa, oedema, haemorrhaging and fibrosis in lung tissues [10] . previous studies on rhesus macaques have shown that histological lesions induced by mers-cov infection were limited to the lungs, which exhibited interstitial pneumonia with a thickening of alveolar septa caused by oedema and fibrin accumulation, and small to moderate numbers of macrophages and even fewer neutrophils. in addition, alveoli tissue samples contained moderate numbers of pulmonary macrophages and neutrophils [17] . previous studies on common marmosets infected with mers also showed that marmosets developed a progressive severe pneumonia or interstitial lymphohistiocytic pneumonia, exhibiting hypoproteinemia consistent with high protein pulmonary effusions resulting from alveolar oedema and interstitial lymphohistiocytic pneumonia with type ii pneumocyte and bronchial associated lymphoid tissue hyperplasia [18, 19] . however, rhesus macaque model did not develop breathing abnormalities and showed no-tovery mild radiographic evidence of pneumonia [20] . similar to our study, the common marmoset model of mers-cov infection mimics the acute and severe pathological process, yet the rhesus macaque model represents the mild infection of mers-cov. thus, the nhp models meet different needs of mers-cov researches. fatal human cases of mers-cov infection cause upper respiratory tract illness, severe pneumonia and multiorgan failure. these cases also include exudative-phase diffuse alveolar damage with the denuding of bronchiolar epithelia, the prominent formation of hyaline membranes, alveolar fibrin deposits, type 2 pneumocyte hyperplasia, the occurrence of rare multinucleated syncytial cells, changes in alveolar septa related to oedema and increases in lymphocytes, with fewer plasma cells, neutrophils, and macrophages [21] . these findings provide evidence for the pulmonary tropism of mers-cov infection. the pathological features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov [22] . previous studies that have evaluated fatal human cases of sars--cov have described diffuse alveolar damage at various stages as the most characteristic feature pathology of mers-cov infection of the disease, with sars-cov antigens primarily found in alveolar epithelial cells. in this study, we examined the histopathological features and the inflammatory cell response that occurs in the lungs of rhesus macaques and common marmosets experimentally infected with mers-cov. our findings indicate that inflammatory cells may play a crucial role in fatal mers-cov infections and that the progression of this disease in our animal models may mimic the infection in humans, making these models useful for further study of the pathogenesis, prevention and treatment of mers-cov infections. in this study, we analysed the distribution of mers-cov protein and viral rna in the lungs of rhesus macaques and common marmosets. we found that pneumocytes and inflammatory cells were positive for mers-cov antigens and viral rna. similarly, in the lungs of both species, mers-cov antigens were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, viral rna was distributed in pneumocytes and inflammatory cells, and viral protein were found in type ii pneumocytes and inflammatory cells. based on our observations, we therefore propose that mers-cov may proliferate and spread from the lungs through an inflammatory cell migration pathway. it has been reported that in fatal human cases of mers-cov infection, viral antigens were identified in both unremarkable and necrotic bronchial submucosal glands and in pneumocytes and epithelial syncytial cells. however, macrophages showed no localization with mers-cov antigens in these cases [23] . findings of pulmonary consolidation, diffuse alveolar damage, and pleural effusion are consistent with the clinical features with mers-cov infection [2, 4, 24] . in this study, we also detected mers-cov protein and viral rna in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells. therefore, our models of mers-cov infection using rhesus macaques and common marmosets may be suitable for use in the development of effective medications and prophylactic treatment and may serve as a tool to improve our understanding of the pathogenic process of mers-cov infection. dpp4 is a single-pass type ii transmembrane glycoprotein with a short n-terminal cytoplasmic tail. the structural residues comprising the receptor-binding domain have been defined via the co-crystallization of the mers-cov spike glycoprotein and dpp4. dpp4 is typically found in type i and ii cells and alveolar macrophages. it has only rarely been detected in the surface epithelia of the nasal cavity and has also been found in a subset of mononuclear leukocytes and serous cells from submucosal glands [25] . in fatal human cases, dpp4 has been observed in scattered pneumocytes and syncytial cells, bronchiolar epithelia and endothelia, and alveolar macrophages [23, 26, 27] . in this study, we found that in the lungs of the nhps infected with mers-cov, ddp4 was expressed in type i and ii pneumocytes, bronchial epithelial cells, and inflammatory cells, primarily alveolar macrophages. in conclusion, we established animal models of mers-cov infection using rhesus macaques and common marmosets, which mimic the oncome of mers-cov infection in humans and provide a tool that may help in better understanding the pathogenic process of this disease. they may also be suitable models for aiding in the development of effective medications and prophylactic treatments for mers-cov infections. fouchier ra isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus in 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tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study key: cord-317688-mr851682 authors: oh, myoung-don; park, wan beom; park, sang-won; choe, pyoeng gyun; bang, ji hwan; song, kyoung-ho; kim, eu suk; kim, hong bin; kim, nam joong title: middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea date: 2018-02-27 journal: korean j intern med doi: 10.3904/kjim.2018.031 sha: doc_id: 317688 cord_uid: mr851682 middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in 2012. the 2015 korea outbreak of merscov involved 186 cases, including 38 fatalities. a total of 83% of transmission events were due to five superspreaders, and 44% of the 186 mers cases were the patients who had been exposed in nosocomial transmission at 16 hospitals. the epidemic lasted for 2 months and the government quarantined 16,993 individuals for 14 days to control the outbreak. this outbreak provides a unique opportunity to fill the gap in our knowledge of mers-cov infection. therefore, in this paper, we review the literature on epidemiology, virology, clinical features, and prevention of mers-cov, which were acquired from the 2015 korea outbreak of merscov. middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in september 2012 [1] . since then, as of 31 january 2018, the world health organization (who) has been notified of 2,143 laboratory-confirmed cases of mers-cov infection, including at least 750 deaths, from 27 countries (fig. 1) [2] . most of mers-cov human infections have occurred in the arabian peninsula. saudi arabia reported 1,783 cases, including 726 deaths (fatality rate, 40.7%) [3] . a recent article summarized the current knowledge on the epidemiology, virology, human pathogenesis, clinical management, and prevention of mers-cov infection [4] . another comprehensive review article on risk factors and determinants of transmission of mers-cov from human to human in the community and hospital settings has been published recently [5] . public health england also published an extensive review of literature for clinical decision-making support for treatment of mers-cov patients [6] . the 2015 korea outbreak of mers-cov is the largest outside the arabian peninsula and provides a unique opportunity to fill the gap in our knowledge about mers-cov infection. since it has now been almost 3 years since the 2015 mers outbreak in the republic of korea, we reviewed the literature to summarize the lessons we have learned from the korea outbreak. the korean journal of internal medicine vol. 33, no. 2, march 2018 fields. as of 31 january 2018, a total of 221 articles were retrieved, and 216 of them were written in english. the full texts of the english articles were reviewed and critically assessed. the 2015 korea outbreak of mers-cov resulted in 186 cases including 38 fatalities [7] . the index case was a returning traveler from the middle east. the infection had spread within the hospital, and subsequently to other hospitals because of patient movement, resulting in nosocomial transmission at 16 clinics and hospitals. the epidemic lasted for 2 months, with the government declaring a "virtual" end to the epidemic on july 6, 2015. in order to control the outbreak, the government quarantined 16,993 individuals for 14 days, and the economic loss was estimated at 9.311 trillion korean won (8.5 billion us dollars) [8] . the first patient (index case) with mers-cov infection was a 68-year-old korean man returning from the middle east. he ran a commercial greenhouse business in bahrain. he flew from seoul to bahrain on april 24, 2015 , and traveled through the united arab emirate and the kingdom of saudi arabia, and came back to seoul on may 4, 2015. during his 10-day business trip, he did not visit a camel farm or a hospital. he did not eat camel meat or any other camel products. he did not remember meeting any persons with respiratory symptoms [9] [10] [11] [12] . seven days after returning home, on may 11, he developed fever and myalgia. he visited a local clinic on may 12, 14, and 15. his symptoms, however, did not improve and a new non-productive cough developed on 15th may. he visited pyeongtaek st. mary's (ptsm) hospital (65 km south from seoul), a secondary hospital in gyeonggi province. his chest x-ray showed a consolidation in the upper zone of right lung, and he was admitted to the hospital for pneumonia on may 15. ceftriaxone and amikacin were started, but his pneumonia progressed despite the antibiotic treatment. he was transferred to 2,103 reported to who as of november 17, 2017 republic of korea other countries saudi arabia 12 18 24 30 36 42 48 01 07 13 19 25 31 37 43 49 03 09 15 21 27 33 39 45 51 05 11 17 23 29 35 41 47 01 07 13 19 25 31 37 43 49 03 09 15 21 27 the chain of transmission was identified in all 186 mers patients [13, 14] . of the 86 healthcare institutions that had taken in mers-cov-infected patients, nosocomial transmission occurred in 12 hospitals and four clinics (plus two ambulances) [13, 15, 16] . the epidemiological characteristics are summarized in table 1 [17] . the epidemic curve is shown in fig. 2 . during his admission from may 15 to 17 at ptsm hospital, the first patient was unknowingly spreading mers-cov. patients, visitors, and healthcare workers (hcws) were exposed to mers-cov. therefore, by may 20, when the korea cdc noticed the importation of mers-cov into the republic of korea, individuals staying on the same floor as the first case had already been exposed to mers-cov. as a result, a total of 36 mers cases occurred at ptsm hospital. of them, 26 patients developed symptoms within 14 days after exposure, suggesting they were first-generation cases; the remaining 10 patients may be second-generation cases [7, 10] . the mode of transmission of mers-cov at ptsm hospital remains uncertain. in addition to family members of the first patient, only one patient shared the same room (#8104) with him; all the other cases stayed in other rooms, which were located > 2 m apart from room #8104. all patient rooms were equipped with an air ventilating system except for room #8104. a recent study using a multi-agent modeling framework suggested the transmission occurred via a long-range airborne route or via a combination of a long-range airborne route and direct contact transmission [18] . a chest computed tomography (ct) scan taken in the early stages (day 1 or 2) of illness showed very small, ground glass nodules in the periphery of the lungs, suggesting inhalation of airborne particles [19] . many individuals had been exposed to the superspreader-1 at the er, and contact tracing identified 675 patients, an estimated 683 visitors, and 218 hcws as contacts of superspreader-1. of them, 278 were quarantined and 617 were under active monitoring. as a result of exposure to superspreader-1, 82 had laboratory-confirmed mers-cov infection, and an additional 10 were secondarily infected from them [20, 21] . the index case (superspreader-2) for the daejeon (100 km south from pyeontaek city) outbreak was infected at ptsm hospital. he developed symptoms on may 20 and was admitted to daechung hospital on may 22. as his pneumonia progressed despite the treatments, he was transferred to geonyang hospital on may 28. a total of 25 secondary cases (14 in daechung hospital and 11 in geonyang hospital) were detected. although superspreader-2 caused the two hospital outbreaks, the me11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 dian time of incubation (8.8 days vs. 4.6 days), secondary attack rates (4.7% vs. 3.0%), and case fatality rate (28.6% vs. 63.6%) were different between the two outbreaks. the high fatality rate was associated with pre-existing pneumonia or poor underlying pulmonary function [22] [23] [24] . a 44-year-old male, who had been exposed to the first index case on may 16, developed back pain on may 21. he is a son of the third mers case. his sister had also been exposed to mers epidemiological characteristics have been reported by several groups (table 2) [7, 16, 20, 21, 23, [27] [28] [29] [30] . the defining epidemiological characteristics were superspreading events at hospitals [30] [31] [32] . early superspreading events generated a disproportionately large number of second-ary infections, and the transmission potential decreased sharply in subsequent generations [30, 33] . a total of 83% of transmission events were due to five superspreaders, and 44% of the 186 mers cases were in-patients who had been exposed within the hospitals [7] . the spreaders transmitted mers-cov from days 1 to 11 of their illness (median, 7 days), and the number of patients infected by each spreader ranged from 1 to 84 (interquartile range, 1 to 12) [27] . the median incubation period and serial interval was 7 days and 12.5 days, respectively [7, 34] . r0 ranged from 2.5 to 7.2, higher than the previous estimate of < 1 [35] . it was estimated at 4.04 for the outbreak cluster in ptsm hospital, and 5.0 for the outbreak in smc [36] . a mathematical modeling study demonstrated that although r0 < 1 overall, cluster sizes of over 150 cases are not unexpected for mers-cov infection [37] . compared with the non-spreaders, the spreaders had a higher frequency of fever (≥ 38.5°c) and chest infiltrates in more than three lung zones, and longer non-isolated in-hospital days [27] . the spreaders had higher viral load in sputum samples with the cycle thresholds for the upe and orf1a genes of 22.7 vs. 27.2 and 23.7 vs. 27.9, respectively. the spreaders with more than three transmissions had higher numbers of contacts and er visits [38] . a brief exposure of a 10-minute stay and 2 minutes of talking was enough for the transmission of mers-cov [39] . the whole genome sequences of mers-cov isolated from the korea outbreak were reported [26, [40] [41] [42] [43] . the virus most similar to the korean isolates was a camel virus (camel/riyadh/ry159/2015) that belonged to lineage 5, a recombinant of lineage 3 and 4 [44, 45] . viruses from lineage 5 had been predominant in saudi arabian camels since november 2014, but human viruses of this lineage were reported from february 2015 [44] . a phylogenetic study of spike glycoprotein genes also showed the korean strains were closely related to the viral strains from riyadh, saudi arabia [46] . in a molecular study, 11 of 13 mers-cov genome had an i529t mutation and one d510g mutation in the receptor binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, cd26 [47] . heterogeneity analysis revealed the combined frequency of i529t and d510g was high (87.7%), although the frequency of both mutations varied greatly among specimens [48] . another genome sequence study also reported deletions of 414 and 419 nucleotides between orf5 and the e protein, suggesting that the virus might be defective in its ability to package mers cov [49] . a microevolution study found no evidence of changes in the evolutionary rate [43] . however, whether the transmissibility and virulence of the korean isolates are different from those of the previous isolates from the kingdom of saudi arabia remains to be determined by phenotypic assays. a viral shedding study showed that the copies of mers-cov rna detected by real-time reverse transcription polymerase chain reaction (rrt-pcr) in respiratory samples peaked during week 2, and the median value was 7.21 log 10 in the severe group and 5.54 log 10 in mild cases [50] . the study also showed that viral titers were higher in throat samples than in nasopharyngeal samples. another study also demonstrated higher viral loads were associated with severity and mortality [51] . after mers-cov infection, antibody responses developed by the third week of illness in most patients [52] . seroconversion rates increased with the increase of dis-ease severity. in a serologic study of 42 mers patients, the seroconversion rate was 0% in asymptomatic infection, 60.0% in symptomatic infection without pneumonia, 93.8% in pneumonia without respiratory failure, and 100% in pneumonia with respiratory failure [53] . the serological response remained detectable for > 12 months in all survivors (11/11) who had severe disease. antibody titers were not detectable in four of six patients who had mild pneumonia, suggesting that mers-cov seroepidemiological studies may underestimate the extent of mild and asymptomatic infection [54] . the korean society of infectious diseases compiled the clinical data of the 186 mers patients [55] . the median age was 55 years (range, 16 to 86). the male to female ratio was 3:2. the most common coexisting medical conditions were hypertension (31.7%), diabetes (18.8%), solid organ malignancy (13.4%), and chronic lung disease (10.2%). patients with mers-cov infection developed a spectrum of illnesses, ranging from asymptomatic to fulminant pneumonia with fatal outcome. the respiratory symptoms were similar to other acute viral respiratory infections. at the time of presentation, fever had developed in 81.7%, cough in 56.7%, and sputum in 39.8% of patients. symptoms of upper respiratory-tract infections were infrequent: sore throat was present in 9.1% and rhinorrhea in 1.6% of patients. gastrointestinal symptoms were also observed: diarrhea was present in 19.4%, nausea or vomiting in 14.0%, and abdominal pain in 8.1% of patients. diarrhea may be due to the side effect of lopinavir/ritonavir, as 87% of the patients received antiviral drugs for mers-cov treatment [55] . in the early stages of illness, cough and sputum were not prominent even in patients who later developed overt pneumonia [56] . at admission, 68% (123/180) of patients had abnormalities on chest radiographs, while 80.8% (147/182) of them developed the abnormalities during the course of the disease [55] . sudden progression of pneumonia occurred around day 7 of illness [50, 57] . predictive factors for development of pneumonia included older age, high fever, thrombocytopenia, lymphopenia, c-reactive protein (crp) ≥ 2 mg/dl, and a high viral load www.kjim.org https://doi.org/10.3904/kjim.2018.031 in sputum (threshold cycle value of rrt-pcr < 28.5) [58] . forty-five patients (24.5%) were treated with mechanical ventilation and 15 patients (8.2%) needed extracorporeal membrane oxygenation [55] . the typical course of pneumonia is shown in fig. 3 . acute kidney injury (aki) developed in 14.0% of the patients, 42.7% (44/103) had proteinuria, and 35.0% (36/103) had hematuria. fifteen patients were treated with renal replacement therapy [55] . old age is an independent risk factor for the occurrence of aki [59] . neuromuscular manifestations were not uncommon; hypersomnolence, weakness and tingling in the extremities were reported during the treatment of mers, suggesting guillain-barré syndrome or virus-related sensory neuropathy [60] . serial changes in chest radiographs was reported in five patients [56] . chest ct scans revealed rapidly developed multifocal nodular consolidations with ground-glass opacity halo and mixed consolidation, mainly in the dependent and peripheral areas [61] . there are few laboratory findings specific to mers-cov infection, although monocytosis with normal white blood cell count and low crp level were more common in mers patients at initial presentation [57, 62] . the guidelines for the molecular diagnosis of mers-cov infection have been published by the korean society for laboratory medicine [63, 64] . specimen type and quality is important in the laboratory diagnosis of mers-cov infection. lower respiratory-tract samples, such as sputum and tracheal aspirates, have higher viral loads than upper respiratory-tract samples [50] . however, mers patients may not shed the virus during the early stage of their illness. therefore, initial negative results should not rule out the possibility of mers [65] , and patients suspected of having mers-cov infection should be retested using a lower respiratory-tract sample [63, 66] . when sputum cannot be obtained, throat swabs may be an alternative source of diagnostic samples [50] . special attention should be given to diagnosing mers-cov infection in immunocompromised patients, as they may present with atypical features, such as a longer incubation period, a longer period from initial pcr positivity to symptom onset, and persistent viral shedding [67] . an antiviral treatment guideline has been published by the writing committee with support from the korean society of infectious diseases and the korean society for chemotherapy [68] . the guideline recommended a triple combination regimen of type 1 interferon, ribavirin, and lopinavir/ritonavir for 10 to 14 days. however, the guideline was mostly based on expert opinions and further study for the optimal antiviral treatment in mers is warranted. the median duration of fever was 8 days. the median time to negative conversion of mers-cov in sputum samples by rrt-pcr was 17 days (range, 4 to 45) [55] . the median time from symptom onset to death was 14 days. the case fatality rate was 20.4% (38/186) (fig. 4) . the in-hospital mortality, 7-day mortality, and 28-day (from symptom onset) mortality were 19.4% (36/186), 3.8% (7/186), and 17.7% (33/186), respectively [55] . host factors associated with mortality were old age (> 60 years), smoking history, pre-existing pneumonia, abnormal renal function, and comorbid conditions [24, [69] [70] [71] . low albumin, altered mentality, and high pneumonia severity index score at admission were risk factors for mortality [69] . mers-cov rna in blood samples was detected in 33% (7/21) of patients at presentation, and it was associated with a worse clinical outcome [72] . a shorter incubation period was also associated with an increased risk of death [24, 73] . a pregnant woman infected with mers-cov in her 35 weeks of gestational age developed dyspnea and her chest radiograph showed diffuse infiltrates in the left lower lung zone. she recovered from the infection and delivered a healthy full-term baby without vertical mers-cov transmission [74] . a patient developed organizing pneumonia 7 days after the resolution of mers-cov infection. he was successfully treated with corticosteroids [75] . in a mental health study, 7.6% of 1,656 patients who were quarantined showed symptoms of anxiety, and 6.4% reported feelings of anger during the 2 weeks of quarantine [76] . mental health support, accurate information, and appropriate supplies, including food, clothes, and accommodation, should be provided to isolated persons [76] . a case of possible transfusion-related acute lung injury following transfusion of convalescent plasma was also reported [77] . a comprehensive "mers infection prevention and control guideline for healthcare facilities" was drafted by the guideline development committee with generous support from korean academic societies [78] . the guideline included practical aspects of infection control and prevention based on the experience from the korea outbreak, such as the composition of members for the mers emergency committee, a space plan for an anteroom for donning and doffing, isolation of in-patients and hcws in a hospital affected by an outbreak, and management of the deceased and autopsy [78] . the hemodialysis unit may become an epicenter for mers-cov outbreak because hemodialysis patients must receive renal replacement therapy even during the outbreak, and the risk of exposure to mers-cov continues. during the korea outbreak, a hemodialysis unit was found to have a mers patient, and a total of 104 patients and 18 hcws were exposed to mers-cov. with support from the korean society of nephrology, the dialysis unit could continue to operate while the exposed patients were isolated in the unit. fortunately, no further transmission occurred at the unit [79] . after this successful experience, the society published a clinical practice guideline for hemodialysis facilities dealing with mers patients [80] . the korea outbreak was driven by the superspreaders who visited multiple healthcare facilities; thus, generating a large number of secondary cases. limiting unnecessary contacts with patients with respiratory symptoms in healthcare settings, especially in emergency departments, is of critical importance [33] . mers-cov could survive for longer than 48 hours at 20°c and 40% of relative humidity, suggesting contact or fomite transmission might occur in healthcare settings [81] . mers-cov was detected by rrt-pcr in specimens taken from the medical equipment [82] [83] [84] . mers-cov was also isolated by cell culture of the air and swab samples taken from a mers isolation unit, suggesting extensive contamination of the isolation unit [85, 86] . of the 186 cases, 23% were infected by undocumented contact between cases (i.e., indirect transmission of mers-cov via environmental contact) [87] . therefore, fomites with possible mers-cov contamination should be sanitized, and a minimum room ventilation rate of six air changes per hour should be implemented to minimize recirculation of pathogen-bearing droplets. meticulous environmental cleaning may be important for preventing transmission in healthcare settings. there was a case of a 39-year-old female nurse who was infected with mers-cov during a cardiopulmonary resuscitation lasting 1 hour for a mers patient who had pneumonia and hemoptysis [88] . serological surveillance conducted after the mers outbreak for asymptomatic infection among hcws involved in the direct care of mers patients showed that 0.3% (2/737) of them were mers-cov igg positive by an indirect immunofluorescent assay. among the hcws who did not use appropriate personal protective equipment (ppe), seropositivity was 0.7% (2/294) compared with 0% (0/443) in hcws with appropriate ppe use [89] . another serological survey also showed that none of the 285 hcws were positive for mers-cov immunoglobulin g, although 38.2% (109/285) of the hcws reported experiencing mers-like symptoms while caring for the mers patients [90] . in a third study, 0 of 189 hcws showed seroconversion by a plaque reduction neutralization test, although 20% to 25% of hcws reported mers-like symptoms [91] . during the outbreak in smc, all hcws assigned to mers patients were screened for mers-cov, regardless of the presence or absence of symptoms. of the 591 hcws, three (0.5%) asymptomatic hcws (two nurses and one physician) were found to be mers-cov (+), but they did not transmit the virus to others [21] . in another study, an asymptomatic nurse without ppe contacted 82 hcws, including 33 close contacts who were exposed within 2 m from the index nurse and were not using ppe. none of the exposed hcws were infected [92] . however, the potential for transmission from asymptomatic rrt-pcr positive individuals is still unknown. therefore, asymptomatic hcws who are rrt-pcr-positive for mers-cov should be isolated and should not return to work until two consecutive respiratory-tract samples test negative on rrt-pcr [93] . the government requested the korean society of infectious diseases to participate in the control of the mers outbreak. on june 8, 2015, the society organized the rapid response team, consisting of 15 infectious-disease doctors and two infection-control professionals [94] . critical suggestions to prevent future epidemics were made regarding a rapid alerting system for index cases, provision of a sufficient airborne infection isolathe korean journal of internal medicine vol. 33, no. 2, march 2018 tion facility, education and training of hcws for important infectious diseases, and overcrowding and visitor control in the hospital [95] . the 2015 korea outbreak was the largest outbreak outside of the middle east. researchers had a unique opportunity to compare the nature of mers-cov infection with the middle east experience. the outbreak unveiled the weak points of infrastructure in our medical system, especially of preparedness for emerging global infectious diseases. nosocomial transmission was one of the core features of mers-cov infection. the introspection for loose medical referral systems, overcrowding at the er, a lack of expert resources and infection control infrastructure, and lack of organized preparedness for medical crises prompted the government to reform the healthcare system, and healthcare sectors to invest further in infectious diseases and infection control. although the improvement is still ongoing, the speed and content are still currently insufficient. international cooperation to prepare for and defeat emerging infectious diseases should also be emphasized. lessons we learned from the outbreak are summarized in table 3 . a single, missed case may trigger a huge, nationwide outbreak the first line of defense is not the thermal scanner at the airport. it is doctors in the community clinics/hospitals. due to sudden deterioration of pneumonia around day 7 of illness, patients may visit emergency department, or be admitted to intensive care unit. superspreading events may occur in healthcare settings, especially at the emergency department. patients may transmit mers-cov as early as 2 days after symptom onset. early detection and isolation is of critical importance. aggressive strategy for quarantine maybe necessary, especially when large number of individuals are exposed in the healthcare settings. huge socioeconomic impact with an estimated 8.5 billion us dollars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) [internet]. geneva: world health organization ministry of health kingdom of saudi arabia. mers-cov daily update ministry of health kingdom of saudi arabia, c2014 middle east respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients uk): public health england middle east respiratory syndrome coronavirus outbreak in the republic of korea the korean middle east respiratory syndrome coronavirus outbreak and our responsibility to the global scientific community the clinical and virological features 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asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance [internet]. geneva: world health organization, c2018 collaborative intervention of middle east respiratory syndrome: rapid response team institutional preparedness to prevent future middle east respiratory syndrome coronaviruslike outbreaks in republic of korea no potential conflict of interest relevant to this article was reported. key: cord-287156-3plpi6i9 authors: lassandro, giuseppe; palladino, valentina; amoruso, anna; palmieri, viviana valeria; russo, giovanna; giordano, paola title: children in coronaviruses’ wonderland: what clinicians need to know date: 2020-07-01 journal: mediterr j hematol infect dis doi: 10.4084/mjhid.2020.042 sha: doc_id: 287156 cord_uid: 3plpi6i9 human coronaviruses (hcovs) commonly cause mild upper-respiratory tract illnesses but can lead to more severe and diffusive diseases. a variety of signs and symptoms may be present, and infections can range in severity from the common cold and sore throat to more serious laryngeal or tracheal infections, bronchitis, and pneumonia. among the seven coronaviruses that affect humans (sars)-cov, the middle east respiratory syndrome (mers)-cov, and the most recent coronavirus disease 2019 (covid-19) represent potential life-threatening diseases worldwide. in adults, they may cause severe pneumonia that evolves in respiratory distress syndrome and multiorgan failure with a high mortality rate. children appear to be less susceptible to develop severe clinical disease and present usually with mild and aspecific symptoms similar to other respiratory infections typical of childhood. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. available data on pediatric coronavirus infections are rare and scattered in the literature. the purpose of this review is to provide to clinicians a complete and updated panel useful to recognize and characterize the broad spectrum of clinical manifestations of coronavirus infections in the pediatric age. structural proteins, including the spike (s), envelope (e), membrane (m), nucleocapsid (n), and sometimes a hemagglutinin-esterase protein (he). the he protein binds to specific receptors and guides membrane fusion; the s protein is responsible for cell entry, the m and e proteins mediate viral assembly process, the inner n protein develops ribonucleoprotein complexes binding to viral rna. [1] [2] [3] [4] [5] to date, seven coronaviruses affect humans: in 1960s hcov-229e and hcov-oc43 were firstly reported; 6, 7 hcov-nl63 and hcov-hku1 were discovered subsequently in 2004 and 2005, respectively. 8, 9 additionally, three hcovs responsible for outbreaks involving high case fatality rates have been detected in humans in the last two decades: the severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the new coronavirus disease 2019 (covid-19) ( table 1) . table 1 . principal features of severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the most recent coronavirus disease 2019 (covid19) . classification beta-cov beta-cov beta-cov in several studies a similar prevalence in the detection of hcovs in patients with respiratory symptoms compared to healthy children has been found. [36] [37] [38] [39] moreover, patients with other underlying medical conditions or immunocompromised appear more susceptible to developing severe infections than healthy patients. [40] [41] [42] [43] additionally, human coronaviruses are responsible for other common childhood diseases such as acute otitis media [44] [45] [46] [47] , asthma exacerbations 48, and conjunctivitis 8 . they have also been involved in nosocomial infections, especially in the neonatal intensive care units (nicu). gagneur et al. in a prospective study determined the incidence of hcovrelated respiratory infections in newborns hospitalized in a nicu. among 64 neonates, seven positive nasal samples for hcovs (11%) were detected. all children were symptomatic. oxygen and ventilatory support were frequently needed. 49 sizun et al. evaluated the clinical role of coronaviruses respiratory infections in premature newborns. all premature infants infected had severe respiratory symptoms, including bradycardia, apnea, and hypoxemia, while chest x-ray revealed diffuse infiltrates. 50 it has also been shown that coronavirus infections are not only responsible for respiratory symptoms but can also affect other organs and systems in children. several studies have also reported that respiratory symptoms caused by coronavirus infection may be associated with central nervous system (cns) involvement. hcovs have an intrinsic capacity to affect neurons and diffuse centrifugally from cns via the transneuronal route. 51, 52 among neurological symptoms, febrile seizures, convulsions, loss of consciousness, encephalomyelitis, and encephalitis have been reported. [53] [54] [55] primarily in 1980, the viral genome was detected post-mortem in the cerebrospinal fluid of two patients with multiple sclerosis (ms). 56 subsequently, the hcovs neuroinvasion capacity was confirmed in a large panel of human brain autopsy samples affected by ms and other neurological diseases. 57 58 in 2017, a prospective study on 192 children with febrile seizures demonstrated that coronaviruses were frequently detected. 59 additionally, hcovs have been implicated as possible causes of many gastrointestinal disorders in children, and gastrointestinal symptoms have been reported in several studies in more than 50% of pediatric patients 28, 60, 61 . firstly, hcovs could be associated with neonatal necrotizing enterocolitis 62 all hcovs can also be detected in stool samples of patients affected by gastroenteritis. 60, 66 moreover, most of the hcovs found were coinfections with well-known gastroenteric viruses, including norovirus and rotavirus. hcovs may also be found occasionally in healthy children's stool samples. 67 although hcovs have always been associated with respiratory symptoms, these findings suggest that other systems may also be involved in children. the absence of serious symptoms may not be coupled with serological negativity. therefore, these viruses should be considered in the differential diagnoses of most of the common diseases of childhood. the 2002-2004 severe acute respiratory syndrome outbreak was a viral respiratory illness caused by sars-cov. the outbreak firstly emerged in the southern chinese province of guangdong in november 2002 and 68 then spread to 29 countries with 8,096 people infected and 774 died. 69 the sars global outbreak was contained in july 2003. since 2004, there have not been any known cases of sars reported anywhere in the world. 70 probably, civet cats or bats could be the initial step of the transmission to humans. humans to humans infection occurs by respiratory droplets or direct contact. healthcare or household contacts are critical routes of transmission. 71, 72 sars-cov infection cases were classified by the world health organization (who) into suspected, probable, and confirmed ( table 2) . 73 the median incubation period ranges between 2-11 days. sars causes atypical pneumonia, which may progress to respiratory failure. symptoms include fever, malaise, myalgia, headache, diarrhea, and rigors. adults are more likely to develop severe illness characterized by dyspnea, lymphopenia, acute respiratory distress syndrome (ards), and a fatal clinical course in 10% of cases. the exact number of children affected by sars worldwide is unknown. however, children appear to be less susceptible to sars with a lower incidence of the disease and no reported mortality. the majority of children had documented exposure to adults with sars, usually a family member. most infected children had previously attended school, but the spread of the infection in the school environment has not been demonstrated, and this could probably be linked to lower infectiousness of the virus among children. 74, 75 children have less severe symptoms than adults, and they rarely need intensive care. however, subclinical and asymptomatic infections appear uncommon. most children reported worldwide were healthy, previously and underlying conditions were infrequently reported. [75] [76] [77] usually, children require hospitalization after 3-4 days the onset of symptoms: fever (90-100%), dry cough (43-80%), sore throat (5-30%), rhinorrhea (33-60%), malaise and myalgia (10-40%), headache (14-40%) are common. dyspnea, tachypnea, and febrile seizures are infrequent. aspecific gastrointestinal symptoms, including abdominal pain, appetite lack, vomiting, and diarrhea, have been reported. physical examination at presentation is negative in the majority of children, and chest auscultation does not reveal significant findings. moreover, sometimes crackles or signs of lung consolidation can be detected. as well as the clinical examination, laboratory findings are not specific in children with sars and can be confused with those of other respiratory infections typical of childhood. commonly lymphopenia, the elevation of transaminases, lactic dehydrogenase, and creatine phosphokinase are detected. other hematological abnormalities such as leukopenia, thrombocytopenia, the elevation of d-dimer levels and mildly prolonged activated partial thromboplastin times are also observed. [78] [79] [80] circulating interleukin (il)-1β levels might be increased, resulting in caspase-1-dependent pathway activation responsible for an exaggerated and persistent inflammatory response and the consequent respiratory failure in severe cases. 81 in children, radiological findings are nonspecific and similar to other viral respiratory abnormalities. high fever (>38 °c) and cough or breathing difficulty and one or more of the following exposures during the 10 days prior to onset of symptoms: close contact with a person who is a suspect or probable case of sars cough or breathing difficulty history of travel, to an area with recent local transmission of sars residing in an area with recent local transmission of sars 1) a suspect case with radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome (rds) on chest x-ray (cxr). 2) a suspect case of sars that is positive for sars coronavirus by one or more assays. see use of laboratory methods for sars diagnosis. 3) a suspect case with autopsy findings consistent with the pathology of rds without an identifiable cause. www.mjhid.org mediterr j hematol infect dis 2020; 12; e2020042 commonly, the chest x-ray shows ground-glass opacity or focal consolidation. linear atelectasis and peribronchial thickening have also been reported. computed tomography (ct) shows more extensive airspace consolidation and ground-glass attenuation than chest x-ray, but it is performed in selective cases in pediatric age. [78] [79] [80] 82 usually, the clinical course is less severe in children compared to adults, and few patients require oxygen supplementation and assisted ventilation but preterm newborns, children younger than one year and older than 12 years of age have more severe symptoms and are likely to develop respiratory distress. [78] [79] [80] in pediatric age, sars infection commonly has a "biphasic" pattern. the first stage of the disease is characterized by virus replication and clinically by the onset of symptoms. the second phase is characterized by pulmonary involvement, which is typically less severe in children than in adults. most children will become afebrile within seven days, and they usually do not progress to respiratory distress, the adult third phase, that is only reported in a minimal number of cases, commonly among teenagers. 83, 84 in pregnant women, sars infection is associated with a high incidence of spontaneous miscarriage, prematurity, and intrauterine growth retardation (iugr). the increased morbidities during pregnancy are likely to be due to the hypoxic state and circulatory insufficiency that worsen placental blood flow and cause miscarriage or iugr. significantly, among pregnant women, mortality is 25%. 85 however, perinatal sars infections have not been documented. in none infants born from pregnant women affected, real-time pcr (rt-pcr) assays and viral cultures conducted on neonatal blood, body secretions and amniotic fluid were positive for sars. in infants, no congenital malformations have been reported. however, in premature newborns, severe gastrointestinal complications such as jejunal perforation and necrotizing enterocolitis have been described 86 . however, it is not known if these neonatal morbidities are related to prematurity or if maternal infection is a factor that increases their incidence. it is unclear why children develop a less serious disease than adults. recurrent viral respiratory infections typical of the pediatric age could be helpful to the immune system in promptly recognizing and defeating new viral pathogens. furthermore, the immaturity of the immune system could be protective because the inflammatory cascade that causes respiratory failure in adults is more difficult to activate. additionally, children generally have fewer comorbidities than adults. children recovered quickly from sars. li et al. assessed the radiological and clinical outcomes of fortyseven children with sars after 6 months from diagnosis. all children were asymptomatic while mild pulmonary abnormalities including ground-glass opacities and air trappings were found at ct in sixteen patients. 87 although clinical and laboratory findings of sars are aspecific in children, certain features can be useful to distinguish sars from other respiratory viral infections. children with sars have a lower incidence of rhinorrhea and productive cough and higher incidence of monocytopenia than children with influenza. 88 additionally, serum lactate dehydrogenase in the presence of a low neutrophil count and low serum creatine phosphokinase could be suggestive of sars infection. 89 sars infections in children appear to be a relatively mild and aspecific disease, and the diagnosis should be accompanied by laboratory assessment. although infants and teenagers are more likely to have a worse clinical course, usually, all pediatric patients recover entirely without significant long-term sequelae. the middle east respiratory syndrome (mers) is a viral respiratory infection caused by the mers-coronavirus (mers-cov). the first identified case occurred in 2012 in saudi arabia. 11, 90 subsequently, a total of 2494 confirmed cases of mers, including 858 associated deaths with a case-fatality rate of 34% were reported globally; the majority of these cases were reported from arabian peninsula, and in the middle east. 91 currently, mers is an extremely rare disease: in the last year mers was signaled only in saudi arabia. 92 mers-cov is a zoonotic virus: dromedary camels are the primary reservoir hosts. humans are infected through contact with infected dromedary camels, animal products, or humans, especially among close contact between family members and health care workers. mers-cov infection cases were classified by the who into suspected, probable, and confirmed ( table 3) . 93 usually, the mean incubation period ranges from 2 to 15 days. clinical severity of the disease varies from asymptomatic to fatal forms, and the impact of asymptomatic spread is unclear. the infection can cause severe pneumonia, which may progress to ards, respiratory failure, and death, particularly in older people, immunocompromised patients, and those with chronic diseases. common symptoms include fever, cough, and shortness of breath. gastrointestinal symptoms (including diarrhea, vomiting, abdominal pain), pericarditis, septic shock and disseminated intravascular coagulation have been reported. [94] [95] [96] [97] children appear to be less susceptible to mers-cov infection, and pediatric cases described in the literature are rare with a low proportion (0.1%-4%) of infected children. 98 age hospitalized with acute respiratory symptoms and/or fever. among these, none of 474 children tested resulted positive for mers-cov. 103 in pediatric age, few cases of mers cov infection have been described. most of the children were asymptomatic and positive during routine screening of mers-cov. al-tawfiq et al. reported a total of 31 pediatric mers-cov cases with a mean age of 10 years. overall, 42% were asymptomatic, while in symptomatic cases, fever and mild respiratory symptoms were common. 104 subsequently, alfaraj et al. reported a total of 7 pediatric mers-cov cases with a mean age of 8 years. in this case series, common symptoms were fever (57%), cough (14%), shortness of breath (14%), and gastrointestinal symptoms (28%). two (28.6%) patients had abnormal chest radiographic findings with bilateral infiltration, one (14.3%) required ventilatory support, and two (28.6%) required supplemental oxygen. 99 four with underlying conditions (cystic fibrosis, nephrotic syndrome, craniopharyngioma, and a right ventricular tumor) had a fatal outcome. these children developed a critical form of mers infection complicated by respiratory and multiorgan failure. frequently, clinical examination revealed bilateral rhonchi and crackles while chest x-ray showed diffuse bilateral infiltrates, ground-glass opacification and pleural effusion. [105] [106] [107] [108] [109] thrombocytopenia, leukopenia, increased creatinine and prolonged prothrombin time were the only laboratory findings reported in literature. 99, 105, 106 mers-cov in children is less frequent than adults and appears to be associated with low mortality unless the patients have underlying comorbidities. few cases of mers-cov have been reported during pregnancy. a pregnant woman, aged 39 years, had a stillbirth at approximately five months of gestation 110 and another woman gave birth to a healthy term baby, but she died after delivery. 107 in conclusion, although mers-cov represents a clinical concern for the adult population with a high fatality rate, it remains a sporadic disease in childhood. clinicians should learn to recognize and suspect mers-cov infection, as the symptoms and signs are nonspecific, based on epidemiological criteria to avoid the spread of the disease in patients at higher risk of worse clinical course. the outbreak of covid-19 infection (coronavirus disease 2019; previously 2019-ncov) began in wuhan, hubei, china, in december 2019, which then spread rapidly to other provinces of china and around the world. 111 on january 30, 2020, the who declared the outbreak of a public health emergency of international concern and, on march 11, 2020, a pandemic. 112 as of june 5, 2020, 188 other countries and regions, with more than 6.669.358 confirmed cases, are declared. among the confirmed cases, 2.904.828 are recovered, and 393.205 died. 113 recent genetic analysis suggests the covid-19 emerged from an animal source. the full genome sequences showed high homology between covid-19, bat coronavirus, and pangolin coronavirus, but further genetic study is required. moreover, according to current evidence, the principal route of transmission of covid-19 is from human to human. 114, 115 covid-19 spread between people through respiratory droplets and contact routes. droplet transmission occurs when there is close contact with a person with respiratory symptoms such as coughing or sneezing, who may spread potentially infectious droplets. transmission may also occur by direct contact with infected persons and indirect contact with infected surfaces or objects. covid-19 can persist on inanimate objects for days but can be efficiently inactivated by common disinfectants. airborne transmission may be possible when a high risk of aerosolization procedures are performed, such as endotracheal intubation and bronchoscopy. the virus is also detected in stool specimens, and consequently, the feco-oral transmission is also hypothesized. [116] [117] [118] [119] the high transmissibility of covid-19 may be explained by its demonstrated presence in the upper respiratory tract of asymptomatic or presymptomatic subjects with viral loads comparable to those detected from symptomatic patients. the real proportion of asymptomatic cases is unclear, ranging from 1% to 78% in different studies. transmission from asymptomatic patients infected with covid-19 most likely contributed to the rapid and extensive spread of pandemic but further studies are needed to more accurately estimate the proportion of genuinely asymptomatic cases and their risk of transmission. [120] [121] [122] [123] [124] [125] [126] covid-19 has been reported among all age groups. the median incubation period of covid-19 infection is 4-5 days with a range up to 24 days. 119, 127 covid-19 infection case is classified by the who into suspected, probable, and confirmed ( table 4) . 128 clinical severity of the infection varies, ranging from asymptomatic forms to critical diseases. common symptoms are fever, dry cough, malaise, lethargy, shortness of breath, sore throat, and myalgia. headache, conjunctivitis, productive cough, and diarrhea are also described. mild forms present as a common cold, and severe cases may worsen in pneumonia that may evolve to ards, shock, and multiple organ dysfunction. more severe clinical pictures are associated with stronger immune response and with the production of proinflammatory cytokines, including il-2, il-7, il-10, and tumor necrosis factor-α (tnf-α). adverse outcomes are common in elderly patients and those with underlying diseases. the need for intensive care admission is in 25-30% of patients. the fatality rate is estimated to range between 2 and 3%. [129] [130] [131] [132] [133] about 2% of covid-19 confirmed cases are children. [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] 135 generally, children appear to be less likely to develop a severe form of covid-19 infection, and commonly they have a mild clinical course with a good prognosis. few children may evolve into lower respiratory infections. probable reasons include having an immune system still immature, healthier respiratory tract, and less underlying conditions than adults. 136 most of them have an infected contact history with family members. moreover, children, especially those with asymptomatic or milder form, may represent significant spreaders. pediatric patients appear to be likely as adults to become infected but are less likely to develop symptoms. however, future studies are needed to understand the role of children in the transmission of the virus. [137] [138] [139] current researches show that the median age of infection in pediatric cases is 6-7 years. in symptomatic cases, symptoms are typical of acute respiratory infections and frequently included fever (59%) and cough (46%), which may be accompanied by nasal congestion, runny nose, conjunctivitis, pharyngitis, wheezing, myalgia, and expectoration. few children have an atypical presentation with gastrointestinal manifestations, including nausea, vomiting, and diarrhea. low oxygen saturation of less than 92%, dyspnea, cyanosis, and poor feeding, are less common than adults. among infants, symptoms such as irritability, reduced response, and poor feeding could be the main signs of infection. family clustering occurred for all infected infants. rarely infants require intensive care or mechanical ventilation or have any severe complications. common symptoms of pediatric age are summarized in figure 1 . the majority of children recovers 1-2 weeks after the onset of the disease. regarding biochemical results, leukopenia and lymphopenia are frequent in children. elevation of transaminases, myoglobin, muscle enzymes, and d-dimers might be seen in severe cases. [140] [141] [142] [143] [144] [145] [146] dong et al. reported that 94% of 2143 pediatric patients affected by covid-19 developed an asymptomatic, mild, or moderate form of infection. a severe disease characterized by dyspnea, central cyanosis, and oxygen saturation of less than 92% was reported in 5% of cases. a critical disease characterized by ards and multiple organs failure was reported in less than 1% of cases. 141 the prevalence of severe and critical disease appears higher in younger children, particularly in children aged <1-year-old and in children with underlying diseases. to date, death was an uncommon event reported in one 10month-old infant with intussusception and multiorgan failure and in one 14-year-old boy. 145, 147 other systemic symptoms appear to be related to the infection, but their link has not yet been demonstrated. since the outbreak of the pandemic, a large number of rashes, urticaria, and vasculitis affecting hands and feet of healthy children and adolescents have been reported as well as itching, burning, difficulty in joint movements and pain. 142 recently, the relationship between covid-19 infection and the development of cardiac diseases in children has been hypothesized. belhadjer et al. have reported a large number of febrile children resulted positive for covid-19 admitted in intensive care units for acute heart failure associated with a multisystem inflammatory state. in most of the children, clinical features appeared similar to those of kawasaki syndrome: lasting fever, cutaneous rash, lymphadenopathy, persistent activation of systemic inflammation and positive response to intravenous immunoglobulin. 148 similar clinical features have subsequently been reported in children with covid-19 positive serology. 149, 150 as in covid-19 infection, kawasaki syndrome is triggered by proinflammatory cascade activated primarily by innate immunity response. however, further studies are needed to establish the real pathogenetic relationship between emerging covid-19 and kawasaki-like syndromes. 151 dufort et al. 152 have recently reported the emergence of a multisystem inflammatory syndrome in children in new york state coincidental with widespread sars-cov-2 transmission, which can better clarify the relationship between kawasaki disease and covid-19. among 191 children admitted to the new york hospitals for multisystem inflammatory syndrome in children (mis-c), 95 patients had a laboratory-confirmed acute or recent severe acute respiratory syndrome coronavirus 2 [sars-cov-2] infection. this hyperinflammatory syndrome manifested with dermatologic, mucocutaneous, and gastrointestinal features associated with cardiac dysfunction. of these 95 patients, a total of 36 patients (37%) received a diagnosis of kawasaki's disease or atypical (or incomplete) kawasaki's disease; 7 of the 9 patients with coronary-artery aneurysms also received a diagnosis of kawasaki's disease. 152 covid-19 infection may also trigger the onset of other immune-mediated diseases such as immune thrombocytopenia, [153] [154] [155] [156] evans syndrome, 157 and autoimmune hemolytic anemia. 158 among radiological findings, ground-glass opacity, mono or bilateral infiltrates, mesh shadows, and tiny nodules are frequently detected. in severe cases, radiological alterations are diffused, presenting as a "white lung." however, radiologic evidence of pneumonia might be absent in 15-20% of children. 139, 140, [159] [160] [161] [162] [163] in selected cases, lung ultrasound might be useful in the managing and follow-up of covid-19 infection. this radiological technique can precociously identify abnormalities including small pleural effusion and subpleural consolidation and appear more available then x-ray and ct. [164] [165] clinical examination appears mostly negative for pulmonary signs, and in rare cases, rales and thoracic retractions have been reported. 161 whether pregnant women and children born to affected mothers are more likely to have a worse outcome is currently unclear. maternal-infant vertical transmission has not been documented. amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples from newborns delivered by infected women were tested for covid-19, and all samples tested negative. 166 data on the maternal and perinatal outcomes of pregnant women infected with covid-19 is limited. most pregnant women with covid-19 present with fever and coughing. severe and critical maternal symptomatology have also been reported, but no women died. the most common adverse pregnancy outcome is preterm birth, occurring in 41% of cases while the rate of perinatal death is 7%, including one case of stillbirth and one neonatal death. there is no data on miscarriage for covid-19 occurring during the first trimester. in more than a third of cases, fetal distress and frequent admission neonatal intensive care units have been reported. 166, 167 rarely, cases of covid-19 positivity in newborns have been reported. common symptoms are fever, cough, lethargy, and vomiting milk. mottled skin and moderate respiratory distress presented with tachycardia, tachypnoea, subcostal retractions, and low oxygen saturation are also described in newborn babies. [169] [170] [171] [172] [173] although it can be severe in some cases, compared with sars-cov and mers-cov, covid-19 causes less severe disease in children. a recent meta-analysis shows that children infected with covid-19 have less fever than that other epidemic hcovs. 174 despite the rapid worldwide spread of covid-19 infection, additional data are needed to define the severity of the disease in children. the severity of the symptoms and the mortality rate will be better assessed in the future. differential diagnosis with common viral respiratory infections of childhood, such as influenza virus, adenovirus, respiratory syncytial virus, and metapneumovirus, should be considered. in the diagnosis of suspected cases, epidemiological and clinical criteria must be assessed. 73, 93, 138 rt-pcr represents the gold standard to confirm the diagnosis of hcovs infections performed on samples of respiratory secretions. [175] [176] [177] [178] [179] [180] [181] the viral load is higher in lower respiratory tract secretion samples than in upper respiratory tract samples. therefore, suspected cases resulted in firstly negative could be re-tested with a second swab, better if with a low respiratory sampling is performed as proved for sars and mers infection. 182, 183 currently, few data have been published about the sensitivity and specificity of rt-pcr nasopharyngeal swabs for covid-19. in vitro analyses suggest that the rt-pcr test is highly specific and sensitive. 184 in vivo, sensitivity is estimated to be higher than 70% but seems to be lower for "mild" cases while specificity is close to 100%. 185, 186 accuracy of rt-pcr swabs in clinical practice differs depending on the site and quality of the sample. taking swabs from children may be more difficult given the intrusive nature of the procedure and further reduce the specificity and sensitivity of the test. rt-pcr of bronchoalveolar lavage fluid appears the most accurate technique of virologic confirmation, but it may not always be easily collected in all patients, especially in pediatric age. although a negative test cannot currently rule out the disease, further studies are needed to define the exact specificity and sensitivity of rt-pcr nasopharyngeal swabs. 187, 188 moreover, rt-pcr appears to be useful in virus detection on stool samples. 116 to date, serology is not considered a diagnostic method. although most patients with covid-19 appear positive for immunoglobulin-g (igg) within 19 days while igm reaches a peak 20-22 days after symptom onset, the serological response is not useful for early individuation of positive patients. 189 additionally, numerous cross-reactions occur between covid-19 and common hcovs 190, and protective immunity against covid-19 is not proved. despite its potential role in supporting rt-pcr in the diagnosis of covid-19, the clinical and immunological meaning of serology is still unclear. 191 the spread of the infection can be prevented if children and family members were educated about proper hygienic practices and infection control measures, including regular hand washing, cover the mouth with napkin or towel when coughing or sneezing, avoid crowded places and contact with sick people. children with hcovs should receive early supportive therapy and continuous monitoring. additional oxygen, caloric, and hydro electrolytic support should be performed if necessary. frequent checks of oxygen saturation and hematological, urinary, and biochemical parameters, including liver, kidney, myocardial enzymes, and coagulation parameters should be analyzed. finally, blood gas analysis and radiological diagnostics of the chest should be done when necessary. this strategy could be useful in the prevention of ards, multiorgan failure, and other nosocomial infections possibly treated, if bacterial, with appropriate antibiotics. in critical cases, mechanical ventilation with endotracheal intubation and other more invasive interventions, such as blood purification and extracorporeal membrane oxygenation (emco), should be adopted. additionally, the use of antiviral drugs in children with severe hcovs infections may help to reduce viral load and the duration of symptoms. however, their safety and real effectiveness have not yet been proven. interferon alfa and beta, corticosteroids, lopinavir/ritonavir, and ribavirin, were used in the treatment of sars-cov and mers-cov in adults and children. 75, 76, 78, 192 however, ribavirin can cause hemolytic anemia and liver dysfunction, as well as corticosteroids, increase the risk of iatrogenic immune immunosuppression. 193 to date, there is no evidence regarding the management and treatment of covid-19 infection in children. in addition to supportive therapy, the use of nebulized interferonalpha-2b and oral lopinavir/ritonavir together with corticosteroids for complications and hydroxychloroquine or intravenous immunoglobulin for severe cases have been suggested. 145, 194, recently, a position paper of the italian society of pediatric infectious disease on the treatment of children with covid-19 infection has been published. 195 in asymptomatic or mild cases, only antipyretic therapy is recommended. in severe or critical cases, the use of hydroxychloroquine ± azithromycin or lopinavir/ritonavir must be considered. immunomodulating therapy with methylprednisolone or tocilizumab or anakinra must be considered in case of the simultaneous presence of ards or progressive deterioration of respiratory function, the elevation of proinflammatory biomarkers and an interval of at least seven days from symptoms onset. supportive therapy should include antipyretic therapy, inhalation therapy with topical steroids and/or bronchodilators and venous thromboembolism prophylaxis therapy. [196] [197] [198] [199] [200] [201] [202] [203] discharge from the hospital is recommended when the patient is without fever for almost three days, respiratory symptoms have improved, and rt-pcr samples are negative. 195 conclusions. most cases of hcovs infection in children have clinically mild symptoms and a relatively short time to resolution. children seem to have a better prognosis compared to adults, and death is a sporadic event. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. learning to recognize pediatric clinical presentations often indefinite or similar to other typical infections of this age, allows clinicians to perform a correct and early diagnosis and prevent the spread of infections in the general population. furthermore, the psychological and social impact of the pandemic outbreak should be considered, especially in the pediatric age. moreover, we think it is necessary to implement innovative clinical tools, such as narrative medicine, to recognize the burden of disease in children and caregivers. 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disease 2019 (covid-19) delle fave a. perceived well-being and mental health in haemophilia a narrative approach to describe qol in children with chronic itp. front pediatr itp-qol questionnaire for children with immune thrombocytopenia: italian version validation's key: cord-295633-vkjcheaz authors: hao, xin‐yan; lv, qi; li, feng‐di; xu, yan‐feng; gao, hong title: the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection via an animal nose‐only exposure device date: 2019-10-30 journal: animal model exp med doi: 10.1002/ame2.12088 sha: doc_id: 295633 cord_uid: vkjcheaz background: middle east respiratory syndrome coronavirus (mers‐cov), which is not fully understood in regard to certain transmission routes and pathogenesis and lacks specific therapeutics and vaccines, poses a global threat to public health. methods: to simulate the clinical aerosol transmission route, hdpp4 transgenic mice were infected with mers‐cov by an animal nose‐only exposure device and compared with instillation‐inoculated mice. the challenged mice were observed for 14 consecutive days and necropsied on days 3, 5, 7, and 9 to analyze viral load, histopathology, viral antigen distribution, and cytokines in tissues. results: mers‐cov aerosol‐infected mice with an incubation period of 5‐7 days showed weight loss on days 7‐11, obvious lung lesions on day 7, high viral loads in the lungs on days 3‐9 and in the brain on days 7‐9, and 60% survival. mers‐cov instillation‐inoculated mice exhibited clinical signs on day 1, obvious lung lesions on days 3‐5, continuous weight loss, 0% survival by day 5, and high viral loads in the lungs and brain on days 3‐5. viral antigen and high levels of proinflammatory cytokines and chemokines were detected in the aerosol and instillation groups. disease, lung lesion, and viral replication progressions were slower in the mers‐cov aerosol‐infected mice than in the mers‐cov instillation‐inoculated mice. conclusion: hdpp4 transgenic mice were successfully infected with mers‐cov aerosols via an animal nose‐only exposure device, and aerosol‐ and instillation‐infected mice simulated the clinical symptoms of moderate diffuse interstitial pneumonia. however, the transgenic mice exposed to aerosol mers‐cov developed disease and lung pathology progressions that more closely resembled those observed in humans. middle east respiratory syndrome coronavirus (mers-cov), which was first identified in saudi arabia in 2012 and causes acute respiratory illness, multiorgan failure, shock and even death, is an important highly pathogenic coronavirus that is similar to severe acute respiratory syndrome coronavirus (sars-cov) and produces severe infections with a high mortality rate. [1] [2] [3] at the end of may 2019, there were a total of 2428 laboratory-confirmed cases of mers with 838 associated deaths (case-fatality rate: 34.5%, which is higher than the fatality rate of sars) worldwide according to world health organization (who) statistics. 4 mers cases have been reported in 27 countries, including countries in the middle east, africa, europe, asia, and north america as well as australia, and case numbers continue to increase, posing a global threat to public health. in china, the first patient infected with mers-cov from south korea was diagnosed in may 2015, 5 and it will be extremely important to prevent, control, and treat mers-cov infections during any future outbreaks. hence, effective small animal models are needed to investigate viral pathogenesis and evaluate mers-cov therapeutics and vaccines. nonhuman primate animal models of mers-cov in both rhesus macaques and common marmosets were established in previous reports, 6, 7 however, these models are limited by restricted availability, high costs, expert husbandry requirements, and ethical concerns. 8, 9 traditional small animals such as mice, hamsters, and ferrets cannot be infected with mers-cov owing to absence of the necessary dipeptidyl peptidase 4 (dpp4) receptor that interacts with the receptor binding domain of the mers-cov spike protein (s protein) [10] [11] [12] mers-cov fails to replicate in mice, which are readily available, have a defined genetic background and low cost and are frequently used in infectious disease research, due to variations in the dpp4 receptor. previous studies showed that transgenic mice expressing the human dpp4 (hdpp4) receptor could be infected intranasally with mers-cov and developed acute pneumonia. [13] [14] [15] therefore, hdpp4 transgenic mice were selected for exposure to mers-cov-containing aerosols using an animal nose-only exposure device. there are two modes of mers-cov infection, animal-to-human and human-to-human transmission. 16 some reports have found that airborne transmission via the coughing and sneezing of infected dromedary camels or contact with respiratory secretions and consumption of unsterilized milk from infected camels can significantly increase the risk of mers-cov infection in humans. 17, 18 kim et al 19 discovered extensive viable mers-cov contamination in the air and surrounding environment in mers isolation wards. according to the who, it has been suggested that human-to-human transmission, to a very limited extent, is caused by inhalation of droplets or airborne virus and close contact with patients. 20 the above studies have demonstrated that mers-cov has a risk of aerosol transmission. in addition, aerosol inhalation is the main clinical route of infection for viral respiratory illnesses. there are different clinical presentations in animal models established by different infection routes. comparative studies using approaches with different perspectives will contribute to a deeper understanding of mers. in this work, to simulate the aerosol transmission route for comparison with the instillation route, hdpp4 transgenic mice were exposed to mers-cov aerosols by an animal nose-only exposure device. after infection, we analyzed the mouse characteristics of weight loss, survival, viral replication, tissue pathology, viral antigen distribution, and cytokine and chemokine profiles, which provide additional data to investigate the pathogenesis of mers-cov-induced disease and evaluate relevant therapeutics and vaccines. specific pathogen-free transgenic c57bl/6 mice expressing hdpp4 all animals were fed under absl-3 conditions for 3 days before the start of the study. on two consecutive days prior to infection, each mouse was trained in an animal nose-only aerosol device. infected mice were kept in the absl-3 laboratory throughout the study and observed daily to ensure that they had enough water and food. mers-cov (human betacoronavirus 2cemc/2012, complete genome genbank: jx869059.2) was provided by the ilas, cams. the virus was propagated and expanded in vero e6 cells (american type culture collection, usa) cultured and passaged at 37°c and 5% co 2 by routine methods. purified and concentrated progeny viruses were titrated using vero e6 cell-based infectivity assays, and viral titers are expressed in units of 50% tissue culture infectious dose per 100 microliters (tcid 50 /100 μl). mers-cov stocks at a concentration of 10 6.8 tcid 50 /100 μl were stored at −80°c. an animal nose-only aerosol exposure device (in-tox products) was located in an absl-3 laboratory and comprised a nose-only exposure chamber and nebulizer inside a class ⅱ biological safety cabinet (bsc ⅱ), a control box, mouse restraint tubes, a clean compressed air tank and a vacuum pump ( figure 1 ). the exposure device, which exposed only the mouse nose, generated mers-cov aerosol particles of 1.27 ± 0.61 μm to infect transgenic mice expressing hdpp4 and simulated the natural route of infection. 21 as shown in table 1 , transgenic mice were randomly assigned to an aerosol group, an instillation group, an aerosol control group, and an instillation control group, and the body weight of each mouse was measured on the day of infection (day 0). each group contained 17 mice; five mice in each group were used to analyze clinical symptoms, weight loss and survival, and three mice in each group were randomly chosen for necropsy on days 3, 5, 7, and 9 postinfection. mers-cov virus suspensions (10 6.5 tcid 50 ) and serum-free dulbecco's modified eagle medium (dmem) were separately added to the nebulizer reservoir to infect exposed mice in the aerosol and control aerosol groups, respectively, for 30 minutes. according to the instructions of the exposure device and mouse respiratory rate (25 ml/min per mouse), the nebulizer flow rate was set to 0.24 l/ min, the diluter flow rate was set to 6.8 l/min, and the nebulizer pressure was set to 20 psi. mice were anesthetized with 1.2% tribromoethanol (0.2 ml/10 g of body weight, intramuscular (im)) for intranasal inoculation with 10 6.5 tcid 50 of mers-cov in the instillation group and serum-free dmem in the instillation control group. infected mice were observed for 14 consecutive days to analyze the clinical symptoms of disease, weight change, and survival. the mice were euthanized with 1.2% tribromoethanol (0.2 ml/10 g of body weight, im) when they reached 25% weight loss. aerosol control dmem aerosol to analyze clinical signs, weight loss, and survival 5 a instillation control dmem suspension to analyze clinical signs, weight loss, and survival on days 3, 5, 7, and 9 postinfection, three animals randomly selected from each group underwent necropsy to obtain tissue specimens for assessing viral distribution, associated histopathology, and cytokine levels using quantitative reverse transcription-pcr (qrt-pcr), hematoxylin and eosin (h&e) staining, immunohistochemistry (ihc), and enzyme-linked immunosorbent assay (elisa). total viral rna was extracted from tissues (lungs, brain, kidneys, spleen, liver, heart, and intestine) homogenized using the rneasy control. 23 a standard curve was generated for pcr using 10-10 7 copies of a qualified standard plasmid to calculate copy numbers for each reaction. formalin-fixed lung, brain, and kidney samples were embedded in paraffin wax and sectioned at an approximately 5-μm thickness. deparaffinized and hydrated tissue sections were routinely stained with h&e to examine histopathological changes. immunohistochemical staining was performed to assess the expression of a viral antigen using a rabbit two-step detection kit (zhongshan golden bridge biotechnology co., ltd) with a rabbit polyclonal anti-mers-cov nucleoprotein (np) antibody (sino biological inc). visualization was then performed by dab staining and hematoxylin counterstaining. supernatants of tissue homogenates from infected mice (50 µl) were added to the bottom of an antibody-coated plate. the levels of interleukin (il)-1β, il-6, il-8, il-10, tumor necrosis factor (tnf)-α, interferon (ifn)-γ, and ifn-β were assayed using elisa kits (kete biotechnology co., ltd). chemokine and cytokine concentrations were recorded as pg/ml of homogenate or ng/l of homogenate. data were analyzed using spss 21 or graphpad prism 5.0 software. the experimental results are presented as the mean plus standard deviation (sd). one-way anova was used to assess differences in body weight, viral load, and cytokine levels among different groups. student's t test was performed for two-group comparisons. p < .05 was considered statistically significant. the infected mice in both the aerosol and instillation groups displayed significant clinical symptoms, such as huddling, hunching, ruffled fur, weight loss, and death. there were significant differences in weight change (p < .001) and survival (p < .0001) between the mers-cov infection groups and the control groups. the incubation period, however, was 5-7 days after aerosol infection and 1 day after instillation inoculation. after mers-cov aerosol exposure, hdpp4 transgenic mice showed profound clinical signs on days 5-7, rapid weight loss on days 7-9 and 60% survival by day 11 (acute death or euthanasia at 25% weight loss). the intranasally infected transgenic mice displayed rapid weight loss on days 1-5 and 0% survival by day 5 (acute death or euthanasia at 25% weight loss). there were significant differences in disease progression (p < .01) after challenge between the aerosol group and the instillation group. transgenic hdpp4 mice infected with mers-cov aerosols exhibited milder disease and slower disease progression than did those inoculated intranasally (figure 2a,b) . no obvious abnormalities, including weight loss or signs of clinical illness, were detected in the aerosol control and instillation control groups. there were no significant differences in weight change or survival rates between mice inoculated with dmem in the above two control groups (p > .05; figure 2c ). based on qrt-pcr analyses of tissue rna contents, we identified high viral loads in the lungs and brain in mice and a small amount of viral rna in other tissues after mers-cov infection via the aerosol or instillation route ( figure 3a ,b). however, there were significant differences in the tissue viral loads of infected mice between the two groups (p < .0001). after mers-cov aerosol infection, high viral loads were detected in the lungs at 3-9 days and in the brain at 7-9 days. viral loads were high in the lungs and brain of intranasally infected mice at days 3 and 5. there were significant differences (p < .0001) in the viral loads in the lungs and brain between the two groups at days 3 and 5. the viral loads in the lungs and brains of the mice in the aerosol group were significantly lower than those of the mice in the instillation group. high levels of viral rna accumulated more slowly in the tissues of the mers-cov aerosol-exposed mice than in those of the mice infected intranasally ( figure 3 ). as shown in figure the data are presented as the mean change ± sd for each group (n = 5). mice in the instillation group died acutely or were euthanized when they reached 25% weight loss; these mice had a 0% survival rate by day 5, which produced no results for weight loss on days 7 and 9. a key indicating the color coding for the groups is provided in the figure. *p < .05, **p < .01, ***p < .001, and ****p < .0001 mice in the instillation group died acutely or were euthanized when they researched 25% weight loss; these mice had a 0% survival rate by day 5, so no tissue lesion results were available on days 7 and 9 mice infected with mers-cov via the aerosol inhalation or intranasal instillation route, but no obvious lesions were found in other tissues. there were no abnormalities in the tissues of the normal control group. it was clear that the appearance of the lungs exhibited obvious congestion and dark brown regions on days 7-9 in the aerosol group. the mers-cov-intranasal mice showed gross lung lesions on day 3 and more severe lung lesions on day 5. gross lung lesions developed more slowly and were milder in the aerosol group than in the instillation group ( figure 4a ). microscopically, challenged mice developed moderate acute interstitial pneumonia and brain pathology, but no pathological changes were detected in other tissues in the mice. in the aerosol group, the lungs of the exposed mice showed alveolar septal widening, inflammatory cell infiltration, and vessel dilatation and congestion at 3-9 days, gradual development of severe pathological changes and inflammatory cell infiltration in perivascular regions at 5-9 days, focal hemorrhages at 7-9 days, and an expanded pathology range at day 9 ( figure 4b ). dilatation and congestion of the cerebral vessels were not clearly observed until day 7, and few areas of neuron deformation necrosis were found in the cerebral cortex, hippocampus, and thalamus before day 9 ( figure 3c ). on days 3 and 5 after intranasal infection, we found moderate acute interstitial pneumonia and brain lesions ( figure 4b ,c). tissue lesions, however, were milder in the aerosol group than in the instillation group. furthermore, there were significant differences in the progression of lung and brain lesions in the two infected groups. tissue lesion progression was slower in the aerosol-infected mice than in the instillation-infected mice ( table 2 ). the expression of a mers-cov antigen was primarily evaluated using ihc assays and was found in endothelial cells and alveolar pneumocytes in the lungs and in cerebral cortical neurons, dendrites, axons, microglia and the hippocampus in the brains of aerosol-and instillation-challenged mice but not in control mice ( figure 5a,b) . prominent mers-cov expression was also observed in renal tubular epithelial cells ( figure 5c ). however, there were significant differences in the timing of virus expression in the tissues of the mice postinfection. after mers-cov infection, ihc assays with a rabbit polyclonal anti-mers-cov np antibody found that viral antigens predominantly appeared in tracheal endothelial cells at day 3 postinfection in the lungs of the aerosol-infected mice and in both tracheal endothelial cells and pneumocytes in the lungs of the aerosol-infected mice at 5-9 days; these changes were observed in the lungs of the instillation-infected mice at 3 and 5 days, respectively. in addition, the mers-cov antigen was discovered in the brain and kidneys in the aerosol group at 5-7 days and in the instillation group at 3 and 5 days. based on these results, we concluded that the distribution of the mers-cov antigen in the lungs, brain and kidneys after infection was slower in the aerosol group than in the instillation group. there were significant differences in the level of related proinflammatory cytokine and chemokine profiles, including il-1β, il-6, il-8, il-10, tnf-α, and ifn-γ, between infectious groups (the aerosol group and instillation group) and the control group (p < .05). significantly elevated levels of il-1β, il-6, il-8, il-10, tnf-α, and ifn-γ were discovered in the lungs and brains of mice in the aerosol group with increased cxcl-1 at 3-9 days (p < .05) postchallenge and in those of mice the instillation group at 3 and 5 days postchallenge ( figure 6 ). in the aerosol group, the exposed mice showed peak il-10 and concentration in the lungs and il-10 and cxcl-1 concentrations in the brain at 5-9 days, and peak tnf-α and ifn-γ levels in the lungs and brains with peak il-6 level at a -, no apparent changes; +, mild alveolar septum widening; ++, moderate alveolar septum widening; and +++, severe alveolar septum widening. b -, no apparent changes; +, infiltration of a few interstitial inflammatory cells; and ++, some interstitial inflammatory cell infiltration. c -, no apparent changes; and +, a small amount of exudate in alveoli. d -, no apparent changes; +, mild dilatation and congestion of vessels; and ++, moderate dilatation and congestion of vessels. e -, no apparent changes; and +, mild hemorrhage. f nd, not done. mice in the instillation group died acutely or were euthanized when they reached 25% weight loss, which occurred by day 5. f i g u r e 5 immunohistochemical staining of mouse tissue samples after infection. a, immunohistochemical staining of the lungs of infected mice. b, immunohistochemical staining of the brains of infected mice. c, immunohistochemical staining of the kidneys of infected mice. mice in the instillation group died acutely or were euthanized when they researched 25% weight loss; these mice had a 0% survival rate by day 5, so no tissue lesion results were available on days 7 and 9 7-9 days. after intranasal infection, however, the levels of il-1β, il-6, il-10, tnf-α, and ifn-γ in the lungs and il-6, il-8, il-10, and ifn-γ in the brains peaked at 3-5 days. the secretion of some cytokines and chemokines in the aerosol group was slower than that in the intranasal group (p < .05). the mice in the instillation group died acutely or were euthanized when they researched 25% weight loss; these mice had a 0% survival rate by day 5, so no results were available on days 7 and 9. the results represent the mean ± sd for each group (n = 3). *p < .05, **p < .01, ***p < .001, and ****p < .0001 distribution in tissues between the aerosol-and instillation-challenged mice (table 3) . after mers-cov infection, the disease progression in the mice in the aerosol group was slower than that in the mice in the instillation group. sanders et al showed that virus droplets were deposited and concentrated in the lungs through the respiratory tract of mice inoculated intranasally, resulting in fast disease onset. 29, 30 correspondingly, after instillation infection with mers-cov, we found that mice with a short airway and high concentration of virus deposited in the lungs displayed weight loss at day 1 and lung lesions at day 3, consistent with intranasal mouse models established by adam, agrawal and li et al [31] [32] [33] ; these mice also exhibited 0% survival by day 5. previous studies reported that aerosol particles ≤5 μm penetrated the respiratory tract to reach the alveoli and were diffusely distributed in the lungs. 30, 34 in addition, virus aerosols entered the blood circulation through the alveoli, and other viruses slowly replicated in the lungs after mice inhaled mers-cov-containing aerosols (particle size: 1.27 ± 0.61 μm). compared with instillation-inoculated mice with virus deposition in the lungs, aerosol-exposed mice displayed slower disease progression with an incubation period of 5-7 days, lung lesions on day 7, continuous weight loss on days 7-11, milder clinical signs, and 60% survival on day 11. we found that the progressions of virus replication and lung lesions in challenged mice were slower in the aerosol group than in the instillation group. based on high viral loads in the lungs and brain of challenged mice, which was consistent with previous reports, 35 and acute renal failure in mers patients, we carried out h&e staining to assess histopathological changes and immunohistochemical staining with a specific antibody to further characterize mers-cov expression in the lungs, brain, and kidneys. a relatively high viral load in the lower respiratory tract is associated with severe illness in viral respiratory diseases. 36, 37 at 3-5 days postinfection, mice in the intranasal group, which had high viral loads in the lungs and brain at 3-5 days, exhibited acute interstitial pneumonia and pathological brain changes. in the aerosol group, mice developed acute interstitial pneumonia at 3-9 days and pathological brain changes at 7-9 days, which were caused by high levels of virus rna in the lungs at 3-9 days and in the brain at 7-9 days, respectively. higher virus rna levels in the instillation group might contribute to the more severe high level on days 3 and 5 high level on days 7 to 9 high level on days 3 and 5 histopathology lungs moderate acute interstitial pneumonia on days 3 to 9 moderate acute interstitial pneumonia on days 3 to 5 brain relatively mild brain lesion on days 7 and 9 brain lesions on days 3 and 5 lungs in bronchial endothelial cells on day 3 in both tracheal endothelial cells and alveolar pneumocytes in the lungs on days 5 to 9 in both tracheal endothelial cells and alveolar pneumocytes in the lungs on days 3 and 5 in cerebral cortical neurons, dendrites, axons, glial cells, and the hippocampus on days 5 to 9 in cerebral cortical neurons, dendrites, axons, glial cells, and the hippocampus on days 3 and 5 in renal tubular epithelial cells on days 5 to 9 in renal tubular epithelial cells on days 5 to 9 cytokines and chemokines b high levels on days 3 to 9, including cxcl-1 high levels on days 3 to 5 middle east respiratory syndrome patients exhibit a median incubation period of 5-7 days, with a range of 2-14 days. 41 we discovered that instillation-inoculated mice exhibited clinical signs within 1 day but that the incubation period of aerosol-exposed mice was 5-7 days, which more closely resembled the period observed in humans. mers-cov binds to hdpp4 receptors that are primarily expressed in the lower respiratory tract and alveoli, resulting in a wide range of disease symptoms in patients, from no symptoms to mild respiratory illness or severe acute pneumonia, which rapidly progresses to acute lung damage, multiorgan failure and even death. 42, 43 clinically, chest radiography and chest computed tomography (ct) show no lung lesions in patients in the early stages of illness, but pneumonia is identified during the course of the disease and includes patchy densities, extensive diffuse and focal alveolar space opacities, interstitial infiltrates, and consolidation. 44 pulmonary pathological changes in aerosol-infected mice were similar to those noted in patients with respiratory tract infection. additionally, immunohistochemical staining revealed that a mers-cov antigen was expressed in alveolar pneumocytes and endothelial cells, the brain, and the kidneys in challenged transgenic mice. studies of a fatal case of mers-cov infection evidenced that the expression of a mers-cov antigen was predominantly localized in pneumocytes and endothelial cells, resulting in cell necrosis and pneumocyte damage; however, no viral antigens were detected in other tissues in the fatal case. 47 as demonstrated in previous studies, we also discovered high viral loads, pathological changes and the expression of a mers-cov antigen in the brain of challenged mice; and no brain lesions, but multiorgan damage, were observed in mers patients. 35, 48, 49 zhou et al demonstrated that human dendritic cells and macrophages were permissive to mers-cov replication, indicating that the multiorgan injury induced by mers-cov may be associated with the distribution of the hdpp4 receptor in many cell types that are spread throughout multiple organs. 38 some studies have indicated that mers-cov has cell and tissue tropisms, especially tropisms for pneumocytes and neurons, and synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. 31, 35 the mechanisms underlying the brain lesions and death induced by mers-cov infection in hdpp4 transgenic mice remain complex and complicated and need to be further investigated. hdpp4 transgenic mice were successfully infected with mers-cov aerosols by an animal nose-only exposure device, and aerosol-and instillation-infected mice all simulated the clinical symptoms of moderate diffuse interstitial pneumonia. compared to instillation-infected mice, aerosol-infected mice more closely resembled infected humans in terms of the progression of disease and pathology in the lungs, which provided additional data for studying pathogenesis and evaluating the efficacy of preventive and therapeutic agents for mers-cov. the current work was supported by the national science and technology major projects of infectious disease (grant number 2018zx10734401-011). none. hg was the principal investigator, designed and supervised the study, and wrote the grant application. xyh performed the main experiments. xyh and ql performed the cell experiments. xyh and fdl conducted the animal experiments. yfx completed the pathology experiments. xyh and hg conceived the experiments, analyzed the data and wrote the paper. all authors read and approved the final manuscript. xin-yan hao https://orcid.org/0000-0001-7664-9568 middle east respiratory syndrome: emergence of a pathogenic human coronavirus nurses' experiences of care for patients with middle east 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clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov key: cord-314651-e4uaw5fy authors: zhao, guangyu; jiang, yuting; qiu, hongjie; gao, tongtong; zeng, yang; guo, yan; yu, hong; li, junfeng; kou, zhihua; du, lanying; tan, wenjie; jiang, shibo; sun, shihui; zhou, yusen title: multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus date: 2015-12-23 journal: plos one doi: 10.1371/journal.pone.0145561 sha: doc_id: 314651 cord_uid: e4uaw5fy the middle east respiratory syndrome coronavirus (mers-cov) causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. for the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of mers-cov infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against mers-cov infection. in this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hdpp4), the receptor for mers-cov. after intranasal inoculation with mers-cov, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. in addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. taken together, this study characterizes the tropism of mers-cov upon infection. importantly, this hdpp4-expressing transgenic mouse model will be applicable for studying the pathogenesis of mers-cov infection and investigating the efficacy of vaccines and antiviral agents designed to combat mers-cov infection. all procedures involving animals were approved by the laboratory animal center, state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology iacuc's (the permit number is bime 2014-0017). animal studies were carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals. all experimental operations to mice were performed under sodium pentobarbital anesthesia, and mice were euthanized with overdose inhalational carbon dioxide. all efforts were made to minimize suffering of animals. considering the species differences, the hdpp4-coding sequence was codon-optimized and synthesized (genscript, nanjing, china) according to the mrna sequence (accession number nm_001935.3) and cloned into the multiple cloning site (mcs) of a pcaggs plasmid, leading to strong expression of the gene. the recombinant plasmid pcaggs-hdpp4 contained the hcmv ie enhancer (hcmviee), the cag promoter (chicken β-actin promoter/intron), a codon-optimized open reading frame (orf) of hdpp4 and rabbit β-globin polya ( fig 1a) . expression of the codon-optimized hdpp4 was achieved by transfection of the pcaggs-hdpp4 plasmid into cos-7 cells, followed by confirmation of expression using anti-human cd26 monoclonal antibody (r&d systems, minneapolis, mn, usa). to confirm the binding of hdpp4 to the mers-cov receptor-binding domain (rbd), transfected cos-7 cells were incubated with a recombinant protein expressing the rbd of mers-cov fused to human igg fc (rbd-fc) [18] , followed by addition of dylight 549-conjugated goat anti-human igg to detect binding. the purified 5861bp fragment generated from apal1 digestion of pcaggs-hdpp4 was used as the transgene for injection into the pronuclei of fertilized f1 (c57bl/6 × c57bl/6) mouse eggs to generate transgenic embryos. the mice used in this studywere backcrossed two to three times onto a c57bl/6 background. all animal studies were performed in strict accordance with the recommendations outlined in the guide for the care and use of laboratory animals. genomic dna from each transgenic founder line and from wild-type c57bl/6 mice was isolated from the liver tissue using dnazol reagent (qiagen, valencia, ca, usa) according to the manufacturer`s instructions. the hdpp4 dna copy numbers were determined by quantitative pcr using power sybr 1 green pcr master mix (life technologies, carlsbad, ca, usa). total hdpp4 dna was normalized using a single-copy reference gene (pkd1) as an endogenous control. the primers specific for hdpp4 were forward 5' ctacagctcctggg caacgtgctg 3' and reverse 5' agatgtcgtaactggcggtgtaag 3'. the primers specific for mpkd1 were forward 5' ggctgctgagcgtctggta 3' and reverse 5' ccaggtcctgcgtgtctga 3'. to detect the expression of optimized hdpp4 in the tissues of our transgenic mice model, samples were harvested and stored immediately in rnalater rna stabilization reagent (qiagen, valencia, ca, usa) until total rna was extracted and purified using an rneasy extraction kit (qiagen, valencia, ca, usa). for each sample, 2 μg of total rna was used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr 1 green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundances of hdpp4 in the tissues. the forward and reverse primers for hdpp4 amplicons were as the same as those noted above. the relative amount of hdpp4 in different tissues was obtained by normalizing mrna expression to that of the endogenous control gene gapdh [19] . mers-cov (hcov-emc/2012 strain) was propagated and titered on vero cells in an approved biosafety level 3 laboratory. following intraperitoneal anesthetization with sodium pentobarbital (5 mg/kg of body weight), mice under virus infection were intranasally inoculated with mers-cov (10 4.3 tcid 50 ) in 20 μl dulbecco's modified eagle's medium (dmem), and mice in sham group were treated with the same volume of dmem. mice were monitored for weight loss and survival for 12 days. we have taken special precautions and followed standard guidelines outlined in the guide for the care and use of laboratory animals to ensure that ample food and water as well as sanitary cage conditions with enrichment devices were present in order to maximize the animal's comfort. humane endpoints were used during the survival experiments. after virus infection, mice were closely monitored by daily observation and weighing of trained animal caretaker, and the monitoring will raise to twice per day once mice lost more than 10% of their initial body weight, or exhibited lethargy, ruffled hair coat, or hunched posture. animals were deemed gravely ill and were euthanized by overdose inhalation of carbon dioxide if they lost more than 25% of their weight, or lost ability to ambulate and access food or water. sera were collected on day 5 and inactivated at 56°c for 30 min before the levels of cytokines and chemokines were measured. to assess viral replication and histopathologic damage following mers-cov infection, mice were euthanized with overdose inhalational carbon dioxide, and tissues included lungs, kidneys, livers, spleens, intestines and brains were harvested on indicated time points. the viral rna copies in tissues were determined by qrt-pcr according to the protocol described elsewhere [20] . briefly, total rna was extracted from 20mg of collected tissues using rneasy extraction kits (qiagen, valencia, ca, usa) following the manufacturer's instructions. mers-cov rna was quantified in a 25 μl mixture containing 5 μl rna using the invitrogen superscript™ iii one-step rt-pcr system with platinum 1 taq (life technologies, carlsbad, ca, usa). the primers and probes specific for the upe envelope gene of the mers-cov virus were as follows: forward 5' gcaacgcgcgattcagtt 3'; reverse 5' gcctctac acgggacccata 3'; fluorescence probe /56-fam/ ctcttcacataatcgccccgagctcg cg/36-tamsp/ [21] . mouse gapdh was used as a housekeeping gene control. a standard curve was generated for pcr reaction using 10-10 7 copies of quantified rna transcripts to calculate copy numbers for each reaction. the results were considered positive at c t values below 35 for primer and probe set, and were calculated as viral rna copies per gram of tissues. the tissues of infected mice were harvested aseptically at indicated time points and homogenized in minimal essential medium (mem) plus antibiotics to produce 10% (w/v) suspensions. tissue homogenates were centrifuged and titered on the monolayer of vero cells. the cytopathic effects (cpe) were daily observed under phase-contrast microscropy for 3 days. the viral titer was determined as tcid 50 by cpe-based assay, and calculated using the reed and muench method. the viral titer in tissue was expressed as log 10 tcid 50 /g of tissue. collected tissue samples were immediately fixed in 10% neutral buffered formalin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (h&e) to examine histopathology as described previously [22] . the expression of virus antigens and infiltration of neutrophils and macrophages in organs after mers-cov infection was detected by immunohistochemistry. briefly, formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated using graded alcohols. expression of virus antigen and inflammatory cell infiltration was assessed using rabbit polyclonal antibody to mers-cov nucleoprotein (np) (sino biological inc., beijing, china), neutrophil marker nimp-14, (santa cruz biotechnology, paso robles, ca, usa) and cd68 (abcam, cambridge, ma, usa). the reaction was detected using a standard streptavidin-biotin detection system (beijing zhongshan biotechnology co., ltd., beijing, china) and visualized using dab with hematoxylin counterstaining. cytokines and chemokines in mouse sera were quantified using a commercial milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, usa.). a panel of inflammatory cytokines and chemokines (ifn-γ, ip-10, il-1β, il-17, il-6, tnf-α, gm-csf, kc, mcp-1, mip-1ɑ, mip-1β and rantes) was measured according to the manufacturer's protocols. statistical analyses were performed using graphpad prism version 5.01. to compare the groups in terms of hdpp4 copies, inflammatory cytokine/chemokine levels, and tissue viral rna copies, student's t test with welch's correction was used. the significance between survival curves was analyzed by kaplan-meier survival analysis with log-rank test. p values lower than 0.05 were considered statistically significant. dpp4 is constitutively expressed in human parenchyma cells in tissues including the liver, intestines and kidneys as well as in t and b cells [7] . we developed transgenic mice in which hdpp4 expression was driven by the chicken β-actin promotor in the pcaggs plasmid ( fig 1a) . expression of hdpp4 in vitro was achieved by transfection of the pcaggs-hdpp4 plasmid into cos-7 cells and detected by immunofluorescence analysis. the results showed that hdpp4 was expressed in cos-7 cells and that it effectively bound mers-cov (fig 1b-1e ). after microinjection of linearized dna, founder lines that transferred the hdpp4 gene to their progeny were established. the levels of transgenic dna in the founder lines ranged from 2 to 6 copies per genome, as determined by qpcr (fig 1f) . in addition, the mrna levels of hdpp4 in the trachea, lung, liver, spleen, kidney, intestine and brain in two of the founder lines were measured by qrt-pcr. the data showed that hdpp4 expression was higher in the trachea, lung, spleen, kidney and brain in both lines, and the expressions were relative higher in trachea and kidney in line 4 compared with that in line 2 ( fig 1g) . in order to determine the effective infection of mers-cov in different lines of transgenic mice, 6 mice in each line were infected with mers-cov and the changes of body weight were monitored. the results showed that all mice in line 1, 2 and 3 survived with no significant changes in body weight but all mice in line 4 died by day 10 following mers-cov inoculation (fig 1h) . in order to confirm the virus replications in transgenic mice, three mice of each line were sacrificed on day 5 after mers-cov infection for the detection of viral titer and viral antigen expression in lungs. as expected, compared with high level of lung viral titers in line 4 mice, no viral titer were detected (fig 1i) in lungs of line 1, 2 and 3 mice, which also confirmed by the negative results of viral antigen expression detected by immunohistochemistry in lung (data not shown). wild-type mice, such as balb/c, 129/svev and stat1 mice, are not permissive to mers-cov infection, and syrian hamsters do not support replication of mers-cov [9] [10] [11] [12] . macaques have been confirmed to develop mild to marked interstitial pneumonia upon mers-cov infection [23] , but fail to progress to the extent observed in human subjects. marmoset model, which has the same interaction characteristic between its dpp4 and mers-cov spike protein, was partially lethal and developed a progressive severe pneumonia after mers-cov infection [14] . in this study, we found that hdpp4 transgenic mice infected with mers-cov exhibited decreased activity and significant weight loss from day 6 after infection, and all of the infected mice died by day 10 (fig 2a and 2b) . importantly, symptoms of neural defects with paralysis were observed on day 9 in the infected transgenic mice. compared to the sham-infected group (fig 3a, 3d, 3g and 3j) , the histopathological analysis of the tissues in transgenic mice on days 5 and 9 after mers-cov infection showed the presence of inflammatory tissue damage in the kidney, liver and spleen, with mild inflammatory responses in the lungs but no significant changes in the intestines. on day 5 after inoculation, the hdpp4 transgenic mice exhibited mild inflammation in the lungs with focal exudation and hemorrhage (fig 3b) , and by day 9, the damage was more severe, with evidence of diffused alveolar damage, alveolar septal thickening, hemorrhage and activated macrophage infiltration (fig 3c) . in the kidneys, mild interstitial inflammation with inflammatory cell infiltration was observed in the interstitium and exudates of renal tubules on day 5 (fig 3e) . by day 9, more renal tubular epithelial cells were injured, with evidence of focal hemorrhage in the renal interstitium (fig 3f) . in the liver, mild to moderate liver damage was seen in the transgenic mice infected with mers-cov on day 5, including scattered hepatocyte necrosis and numerous activated kupffer cells and macrophage infiltrates in hepatic sinusoid (fig 3h) , and on day 9, fatty change in hepatocytes with less hepatocyte necrosis was observed (fig 3i) . in spleens, necrosis of splenic cells and increased reticulum cells in red pulp with significant hemosiderin deposition was observed on both day 5 and day 9 (fig 3k and 3l) . typical characteristics of viral encephalitis were observed in the brain at day 9, with perivascular cuffs ( fig 3m) and neuronal cell necrosis in the cerebral cortex (fig 3n) , including hippocampal neurons (fig 3o) . there was no apparent damage in the intestines after mers-cov infection on day 5 or day 9 (data not shown). to evaluate mers-cov infection in the hdpp4 transgenic mice, distribution of the viral antigen np protein was assessed in mouse tissues by immunohistochemical staining. the results showed that type i and type ii pneumocytes in the lungs and renal tubular epithelial cells in the kidneys expressed viral antigen on day 5 after mers-cov infection and that the expression was more abundant on day 9, and no viral antigen was detected in sham-infected group ( fig 4a-4f) . strong viral antigen expression was evident not only in neuronal cell bodies, but also in dendrites, axons and some microglia in the cerebral cortex (fig 4h) including the hippocampus (fig 4i) . no viral antigen expression was observed in the brains of mice in the shaminfected group (fig 4g) . viral rna copies in lung and brain were detectable on day 5 postinfection and increased to a higher level on day 9 (fig 4j) . lower level of viral rna copies can be also detected in kidney collected on day 9. in order to further confirm the effective replication of mers-cov, the dynamic changes of viral titer in organs were detected. the results showed that on day 3 after infection, only lower viral titer was detected in lung and not detectable in other organs. however, on day 5, viral titer was also detected in brain. on day 7 and day 9, increased viral titers were detected in both lung and brain, and lower level viral titer in kidney was detectable (fig 4k) . these results indicate that the transgenic mice had been successfully infected and that mers-cov exhibited cell and tissue tropism especially for pneumocytes and neurons. furthermore, synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. to date, only limited data on the immune response of mers patients are available [24] . because of the clinical similarity between the symptoms of mers-cov and those of patients infected with sars-cov [1] , the aberrant immune response may be related to the pathogenesis of mers-cov infection. in our mouse model, an aberrant inflammatory response was confirmed by infiltration of neutrophils in the lung, liver and spleen (fig 5a-5f ) and macrophages in the lung, liver and kidney (fig 5g-5l ). in addition, deposition of hemosiderin in the spleen (fig 3k and 3l ) indicated an increase in activated macrophages, and a systemic inflammatory response after virus infection was demonstrated by a significant increase in cytokines such as ifn-γ, ip-10, and il-17 in the serum on day 5 after mers-cov infection (fig 5m) . although several species, such as rhesus macaques and common marmosets are reported to be sensitive to mers-cov infection and to develop mild to marked broncho-interstitial pneumonia, small animal models are urgently needed to study the pathogenesis of this disease and evaluate the effects of vaccines and antiviral agents. a mers-cov-infection mouse model was generated by transducing mice with a recombinant non-replicating adenovirus expressing hdpp4; however, while the mice exhibit viral antigen expression and progress to interstitial pneumonia, there is no mortality after virus infection [15] . although a transgenic mouse model expressing human dpp4 was also established, and its immune response was studied after infection with mers-cov [16] , the transgenic mice in the study died on day 6 with only progressive pneumonia and mild perivascular cuffing in brain, and no neurological disorder or other multi-organ damage was observed. different from the aforementioned transgenic mice, our hdpp4 transgenic mice experienced a longer incubation period post-infection and developed progressive pneumonia and neurological disorders accompanied by histological damage to the lungs, brain, spleen and liver, which more closely resembles the clinical cases. the results from this study indicate that mers-cov infection was lethal in hdpp4 transgenic mice with higher transgene copy number, while lines with lower levels of hdpp4 expressed especially in trachea had no significant clinical symptoms (data not shown), suggesting that the copy number of the transgene was closely related to the efficiency of mers-cov infection. although the expression level of optimized hdpp4 was not highest in the brains, the tissues had higher viral titers following mers-cov infection, revealing the tropism of mers-cov for both lungs and brains, tissues in which pneumocytes and neurons may be the main target cells (fig 4) . however, mers-cov infection in brain via blood or olfactory nerves needs to be further studied due to the strong expression of viral antigens in dendrites and axons. although few autopsy reports exist on fatal mers-cov cases and atypical pneumonia and respiratory failure are suspected the causes of death [24] , other types of extrapulmonary organ dysfunction have also been documented in mers critically ill patients with abnormal clinical manifestations [25] [26] [27] . for example, acute renal failure was described in a number of mers cases [28] [29] [30] [31] [32] . in addition, renal epithelial cells may produce almost 1000-fold more infectious mers-cov progeny than bronchial epithelial cells [33] . more recently, severe neurologic syndrome including altered level of consciousness from confusion to coma, ataxia and focal motor deficit was also reported in three critically cases by balkhy h et al [4] . according to the neurologic manifestations and distinct imaging patterns, an important question was raised on the pathogenic mechanisms that underlie the occurrence of neurologic injury in patients. it has been studied that corona virus such as sars-cov are generally known for causing respiratory illness, and the strong tropism of sars-cov to cns and causing neuronal injury had also been demonstrated both in clinical and experimental studies [34] [35] [36] [37] [38] , which suggested the possible relations of mers-cov infection with brain injury. as to the extensive expression of hdpp4 in the lung, kidney, placenta, liver, skeletal muscle, brain, endothelium, pancreasour and t cell in human, our globally expressed hdpp4 transgenic mouse manifested with multiorgan damage infected with mers-cov could be a suitable model for the pathogenic mechanisms study. systemic inflammation is believed to be a primary reason for the severe outcome in mers--cov infections [14, 24] . although the transgenic mice model died after mers-cov infection with multi-organ damages, it is still uncertain whether these mice are mainly dying from lung damage. in our transgenic mice, aberrant immune response such as elevated ip-10, ifn-γ and il-17, which are closely related to acute virally-mediated lung injury [39] [40] [41] [42] , was also a specific manifestation after mers-cov infection. so, as to the mechanism of lethal which was due to the virus replication directly or induced by the aberrant inflammatory response need to be further studied. the mechanism of mers-cov infection caused death is complex and complicated. aberrant immune response after mers-cov infection may be one of main reasons which need to be further studied. we have added more discussions on this issue in the revised manuscript. in summary, the transgenic mouse model developed in this study was efficiently infected by mers-cov and exhibited severe acute respiratory injury and considerable extrapulmonary organ damage. this model will be useful for studying the pathogenesis of mers-cov infection and evaluating the efficacy of mers vaccines and therapeutic agents. molecular pathology of emerging coronavirus infections state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hospital outbreak of middle east respiratory syndrome coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the multifunctional or moonlighting protein cd26/dppiv middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd wild-type and innate immune-deficient mice are not susceptible to the middle 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ill patients with middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection latest outbreak news from promed-mail: novel coronavirus-middle east clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection possible central nervous system infection by sars coronavirus detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis multiple organ infection and the pathogenesis of sars pathology and pathogenesis of severe acute respiratory syndrome susceptibility of human and rat neural cell lines to infection by sars-coronavirus early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus severe acute respiratory syndrome in children interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome therapy of h7n9 pneumonia: current perspectives key: cord-287761-73qgx58i authors: aly, mahmoud; elrobh, mohamed; alzayer, maha; aljuhani, sameera; balkhy, hanan title: occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update date: 2017-10-13 journal: plos one doi: 10.1371/journal.pone.0183850 sha: doc_id: 287761 cord_uid: 73qgx58i the emergence of the middle east respiratory syndrome coronavirus (mers-cov) infections has become a global issue of dire concerns. mers-cov infections have been identified in many countries all over the world whereas high level occurrences have been documented in the middle east and korea. mers-cov is mainly spreading across the geographical region of the middle east, especially in the arabian peninsula, while some imported sporadic cases were reported from the europe, north america, africa, and lately asia. the prevalence of mers-cov infections across the gulf corporation council (gcc) countries still remains unclear. therefore, the objective of the current study was to report the prevalence of mers-cov in the gcc countries and to also elucidate on its demographics in the arabian peninsula. to date, the world health organization (who) has reported 1,797 laboratory-confirmed cases of mers-cov infection since june 2012, involving 687 deaths in 27 different countries worldwide. within a time span of 4 years from june 2012 to july 2016, we collect samples form mers-cov infected individuals from national guard hospital, riyadh, and ministry of health saudi arabia and other gcc countries. our data comprise a total of 1550 cases (67.1% male and 32.9% female). the age-specific prevalence and distribution of mers-cov was as follow: <20 yrs (36 cases: 3.28%), 20–39 yrs (331 cases: 30.15%), 40–59 yrs (314 cases: 28.60%), and the highest-risk elderly group aged ≥60 yrs (417 cases: 37.98%). the case distribution among gcc countries was as follows: saudi arabia (1441 cases: 93%), kuwait (4 cases: 0.3%), bahrain (1 case: 0.1%), oman (8 cases: 0.5%), qatar (16 cases: 1.0%), and united arab emirates (80 cases: 5.2%). thus, mers-cov was found to be more prevalent in saudi arabia especially in riyadh, where 756 cases (52.4%) were the worst hit area of the country identified, followed by the western region makkah where 298 cases (20.6%) were recorded. this prevalence update indicates that the arabian peninsula, particularly saudi arabia, is the hardest hit region regarding the emerging mers-cov infections worldwide. gcc countries including saudi arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. the emergence of mers-cov dates back to july 2012 when an elderly patient of age 60 years died from an acute pneumonia in saudi arabia, and a new coronavirus strain was isolated from his lung tissue [1] . another case of acute respiratory disease was diagnosed in a 49-year old male in london who was from qatar and a new strain of coronavirus was isolated from this patient as well [2] . shortly after, the entire genome of the new coronavirus was sequenced and deposited in the genebank database under the number jx869059, kc164505.2. the phylogenetic analysis of the new virus genome revealed that homology of the nucleotide sequence between two cases was 99.5% and the isolates were closely related to bat coronavirus (bat-cov) which belongs to group 2c of β-coronavirus [3] . according to the recommendations by the international committee on taxonomy of viruses (ictv), the new coronavirus was named as 'middle east respiratory syndrome coronavirus' (mers-cov) [4] . although, initially reported from the middle east, mers-cov exported cases have also been observed worldwide. with regard to viral origin and transmission, the first case of mers-cov infection did not relate it to any particular contact with animals before the disease onset; however, other studies did link it to dromedary camels [5] [6] [7] [8] . beta-coronaviruses are strongly associated with bats serving as reservoir host, particularly, the african neoromicia bats were speculated to be the natural reservoir of mers-cov [9] [10] [11] [12] . notably, serological evidence suggests that mers-cov has been in the circulation for at least 2-3 decades in dromedary camels [13] . given that, the ancestral origin of mers-cov links it to african bats whereas, dromedary camels have been functioning as an intermediate host for this virus for a significantly long period of time [14] [15] [16] . health facilities, hospitals and households with mers patients are considered to be the epidemic centers of mers-cov outbreaks. mers-cov is mainly spreading across the geographical region of the middle east while only sporadic cases are reported in the europe, north america, africa, and lately asia. this may be because of the widespread population of dromedary camels in the middle east; however, the scientific proof of evidence that camel farms are a potential source of mers-cov infections still remains to be established. besides, the typical seasonality pattern is not seen in case of mers-cov infections and only one report links it to camel breeding season. the modes of mers-cov transmission by droplet, contact, or airborne are not yet confirmed as well and thus its transmission among animals and from animals to human and human to human remains unclear. there is also no documentation available regarding mers-cov transmission during airplane flights. therefore, standard infection prevention and control procedures are followed including droplet and airborne precautions. the viral incubation period is from 2 days to 2 weeks. the viral cytopathic effects clearly show prominent syncytium formation in humans as well as non-human primates. mers-cov targets directly the lower respiratory tract (pneumocytes) in dromedary camels and continues to replicate preferentially in the airway cells of the upper respiratory tract. the clinical manifestations of mers-cov infections represent a wide spectrum ranging from asymptomatic cases to the ones with severe respiratory indexes. according to the who, mers-cov infection is an acute respiratory infection involving pyrexia of 38˚c or more, cough with radiologic pulmonary presentation and also the history of the patients originating from or travel to the arabian peninsula and its neighboring countries within 10 days of symptoms. mers-cov cases have been identified as both community-and hospital-acquired, mainly among the aged population and in patients with multiple comorbidities such as acute pneumonia, upper respiratory tract infections, influenza-like illness, or asymptomatic infection(s) in children and immunocompromised hosts. moreover, the common extra-pulmonary symptoms include diarrhea and acute renal failure. the early clinical diagnostic changes include the impaired liver and renal functions, lymphopenia, leukopenia and thrombocytopenia whereas leukocytosis, and neutrophilia are linked to progressive infections. the gold standard for diagnosis is detection of viral rna by rt-pcr in compliance with the who guidelines for positive case criteria. the virus is found to be present in different diagnostic specimens such as the lower respiratory tract, sputum, endotracheal aspirate, bronchoalveolar lavage; upper respiratory tract, nasal or nasopharyngeal swabs, urine, feces, and blood. nevertheless, positive biopsy and autopsy tissue specimens still remain to be reported. direct or indirect contact seems to explain a part of the transmission kinetics observed between dromedary camels and humans. in the general population, transmission is rather inefficient (r0<0.7) and it was reported that mers-cov mortality rate is 35% [17] . however, once the virus is introduced into hospital setting with large numbers of susceptible patients at risk, the virus appears to be transmitted very efficiently among such vulnerable host populations. regarding viral evolution and natural reservoir, dromedary camels are the natural reservoir for mers-cov and given the multitude of different clades found in both dromedary camels and human outbreaks, it appears that virus evolution takes place in the reservoir host rather than in humans. the study objective was to report the prevalence of mers-cov infections in the gcc countries and to also investigate its demographics in the arabian peninsula. the data for the last 4 years were collected form the king abdulaziz medical city, riyadh, ksa. further data were collected from who s1 table and also from ministry of health portals of the gcc countries as follows: bahrain http://www.moh.gov.bh/, kuwait www.moh.gov.kw, oman www.moh.gov.om, qatar www.moph.gov.qa, united arab emirates http://www.moh. gov.ae/, and saudi arabia http://www.moh.gov.sa/. we also consulted the gcc countries' reports and websites for the incidence of mers-cov infections between june 2012 and july 2016. furthermore, we searched pubmed database for articles form gcc countries reporting mers-cov infections. epidemiological data including age, sex, symptoms, date of onset, and date of sampling were collected and entered into excel worksheets. descriptive analysis, frequencies and percentages were calculated using spss vr. 20 statistical software. between june 2012 and july 2016, a total of 1797 confirmed mers-cov cases were reported worldwide with a mortality rate of 38.2% (n = 687). the regional distributions of mers-cov were as follows: middle east had the highest number cases (88.4%), followed by asia (10.7%), europe (0.8%) and usa with only 2 cases officially reported (0.1%) the data are summarized in table 1 and fig 1a. one hundred fifty five patients out of the total 1797 confirmed cases (8.6%), reported their exposure to animals, of which, 130 out of 155 cases (83.9%) were exposed to camels, while 25 out of 155 (16.1%) stated exposure to other animals including sheep, cows and poultry ( table 2 ). despite the fact that 674 out of 1797 mers-cov cases (37.5%) were health careassociated infections and 284 out of 1797 cases (15.8%) involved contact with an infected family member, 147 cases (8.2%) were still reported with no exposure to any of the above. the exposure data were found missing for the remaining 537 (29.9%) patients. the distributions of mers-cov infections among 6 gulf countries are illustrated in table 3 and fig 1b. the majority of mers-cov infections (93%) reported between the time period from june 2012 to july 2016, were from saudi arabia. while, the remaining 5 gcc countries contributed only 7% of the cases with the distributions as follows: united arab emirates (5.0%), qatar (1.0%), oman (0.5%), kuwait (0.2%) and bahrain (0.06%). gender analysis shown in table 3 reveals that 60% of the patients were male and 31% were female. moreover, saudi arabia. next, we sought out the detailed demographic distributions of reported cases among 14 governorates in saudi arabia. as shown in table 4 and fig 1c, most of the cases (52%) were reported in al riyadh region, making it the worst hit area in the country followed by makkah (20%), ash sharqiyah (11%), al madinah (4%) and najran (3%). the remaining 9 regions together contributed to 5% of the cases. to date, cases are still logged from ksa and newly-diagnosed positive cases are on the rise. bahrain. to date, there was only one case reported from bahrain in manama region. herein, a 61-year-old saudi male was admitted on the 29 th of march, 2016 to a health care facility in bahrain for an unrelated medical condition. this person was later on tested as positive for mers-cov ( table 1) . state of kuwait. according to kuwait ministry of health, a total of 4 cases were confirmed as mers-cov infections. the first case was reported form the capital (kuwait city) on and an 85-year-old female from abu dhabi were traced to another confirmed mers-cov patient with no history of exposure to camels or other risk factors. finally, a 37-year-old expat male from abu dhabi developed symptoms on the 9 th of june, 2016 and was later tested positive for mers-cov. finally, we searched for the pattern of mers-cov infections over the months in order to identify seasonality relationship. as shown in fig 2, the average of the reported cases during this 4 years period shows that 60-80 cases are reported in the period between february and may, while about 90-100 are reported in august and september. in addition, a low point of infection occurs in the period of october-january and another one in june. the highest number of cases were reported overall during the summer time. over the past 4 years or so, increasing numbers of mers-cov infections have been reported from the middle eastern region [1] . herein, we present a prevalence update on the current status of mers-cov infections in the gcc countries. the data collected over a time period from june 2012 and july 2016 show that the highest number of cases (1441) were reported from saudi arabia (93%) among a total of 1797 cases reported worldwide. overall, a total of 1550 cases were reported only from the gcc region. the saudi arabian capital city of riyadh with 756/1441 (52.4%) cases remained the hardest hit area for mers-cov outbreaks. the incidence of mers-cov infections was found to be highest among the elderly population aged 60 yrs or above. moreover, the gender analysis showed that there were twice more number of males infected (871/1317) than females (446/1317). there is no evidence that mers-cov has gender predisposition [18] . the observed gender-related rates could be simply due to the higher probability of male exposure to camel population than females in the region [18, 19] . furthermore, over one third (37.5%) of mers-cov patients received intensive care among all hospitalized cases [20] . one could argue that the hot climate shared by arabian peninsula and sub-saharan african region could contribute to the spread of mers-cov infections across these geographical regions. although lesser in number, there are still numerous mers-cov infections recorded in saudi arabia during the winter time as compared with summer. the seasonality pattern analysis identifies a 2-phase annual cycle wherein the outbreaks occur during the winter and summer months. altogether, the summer time represents the peak season for mers-cov infections and transmission. the evolutionarily related bat virus might have undergone modifications and adaptation in order to be able to successfully infect and multiply in the camel as an intermediate host before transmission to the human host [21] . although, camel is a well-known animal that is widely colonized in the gulf region, it is also reared and maintained in other parts of the world. we speculate that there might be certain conditions or factors involved with regard to camel herding and shepherding exclusively in saudi arabia that would have facilitated and contributed to the survival of pathogen and fast spread of mers-cov infections from camels to humans across all over the country. there are some reports showed that human consumption of unpasteurized camel milk and or other camel products maybe a reason for the zoonotic transmission of mers-cov in the region [22] [23] [24] . on the other hand, there is strong evidence that mers-cov has been circulating in the dromedary camel population for more than 2 decades [25] . yet, the reason that first human infected case was identified in 2012 remains unclear. we found that mortality rates were higher among the elderly group for both genders which was also concordant with a previous study [26] . the possible explanation for the enhanced mortality in aged patients could be the presence of senescence-associated immune vulnerability in these individuals and suboptimal immune reactivity following a systemic challenge by exposure to mers-cov natural infection. other gulf countries show a few sporadic cases which may be due to the missing data that still have to be set straight or it could possibly be due to small size of camel populations is wide spread across vast geographical region. there may be still other factors involved that remain hidden at present but contribute significantly to the survival, transmission and pathogenesis of this relatively newly identified pathogen in this region of the world. notably, it appears as if coronaviruses are able to cause serious viral infections when transferred from their reservoir (wild bat) host to the human host as observed previously for ebola virus as well which was transmitted from wild bats to humans in africa. actually, many of mers-cov cases are initiated in rural areas and following hospitalization, further cases were reported. the majority of the mers-cov outbreak cases took place at health facilities; index cases are very crucial and they raise the question of the route of transmission of this zoonotic virus. this warrants caution that strict healthcare protocols and guidelines need to be followed and practiced by health care personnel in order to prevent the new outbreaks of mers-cov. in conclusion, mers-cov infections were reported to occur in saudi arabia during the whole year whereas the incidence of human outbreaks peaked in winter and summer months. the disease incidence was also highest among the elderly population aged 60 yrs and above. besides, the fact that majority of these cases were due to human to human interaction i.e. especially among the hospitalized icu patients and not due to camel to human transmission, the local health sectors need to be made aware to mandate implementation of effective control strategies and stringent compliance with better standards of health and hygiene nationwide. despite all whistle blowing efforts aimed at raising awareness of the magnitude of the problem at home, further efforts are still needed for proper treatment and care of mers-cov-infected patients in this country. last but not least, the availability detailed reports of each and every case of mers-cov infection globally, and the gcc region particularly will provide valuable information to the scientific community that may be used to track, contain, and eradicate this disease more effectively. supporting information s1 table. isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate infection control and mers-cov in health-care workers mers coronovirus has probably been present in bats for many years, research shows link to mers virus underscores bats' puzzling threat genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus neutralizing antibodies in camels animal models of middle east respiratory syndrome coronavirus infection mers-cov an emerging viral zoonotic disease: three years after and counting. recent pat antiinfect drug discov middle east respiratory syndrome prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis middle east respiratory syndrome coronavirus: review of the current situation in the world mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir human infection with mers coronavirus after exposure to infected camels, saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels mers coronaviruses in dromedary camels a lesson learned from middle east respiratory syndrome (mers) in saudi arabia the authors thank the kaimrc members and infectious disease research unit for their help and support. this study was approved by the irb and supported by funds from king abdullah international medical research center kaimrc (grant #rc15/128). conceptualization: mahmoud aly. key: cord-323125-qtlevnbt authors: al hosani, farida ismail; kim, lindsay; khudhair, ahmed; pham, huong; al mulla, mariam; al bandar, zyad; pradeep, krishna; elkheir, kheir abou; weber, stefan; khoury, mary; donnelly, george; younis, naima; el saleh, feda; abdalla, muna; imambaccus, hala; haynes, lia m; thornburg, natalie j; harcourt, jennifer l; miao, congrong; tamin, azaibi; hall, aron j; russell, elizabeth s; harris, aaron m; kiebler, craig; mir, roger a; pringle, kimberly; alami, negar n; abedi, glen r; gerber, susan i title: serologic follow-up of middle east respiratory syndrome coronavirus cases and contacts—abu dhabi, united arab emirates date: 2019-02-01 journal: clin infect dis doi: 10.1093/cid/ciy503 sha: doc_id: 323125 cord_uid: qtlevnbt background: although there is evidence of person-to-person transmission of middle east respiratory syndrome coronavirus (mers-cov) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. methods: a seroepidemiological investigation was conducted among mers-cov case patients (cases) and their household contacts to investigate transmission risk in abu dhabi, united arab emirates. cases diagnosed between 1 january 2013 and 9 may 2014 and their household contacts were approached for enrollment. demographic, clinical, and exposure history data were collected. sera were screened by mers-cov nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. results: thirty-one of 34 (91%) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. mers-cov antibodies were detected in 13 of 24 (54%) case patients with available sera, including 1 severely symptomatic, 9 mildly symptomatic, and 3 asymptomatic case patients. no serologic evidence of mers-cov transmission was found among 105 household contacts with available sera. conclusions: transmission of mers-cov was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. these results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission. although there is evidence of person-to-person transmission in household and healthcare settings [10] [11] [12] [13] [14] , more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. these data would be of importance to the public health response given that approximately 25% of confirmed mers-cov cases reported to the world health organization have been described as mildly symptomatic or asymptomatic [15] . during 1 january 2013-9 may 2014, the department of health-abu dhabi (doh) investigated 65 laboratory-confirmed cases and conducted extensive contact investigations in both household and healthcare settings [5] . through these investigations, 72% of the laboratory-confirmed cases reported no symptoms or mild illness [5] . contacts of case patients were tested by diagnostic pcr assays; however, results could include false negatives due to the 14-day incubation period. in this investigation, we use serological detection of mers-cov antibodies to evaluate if asymptomatic or mildly ill case patients had detectable mers-cov antibodies, estimate transmission rates from known cases to their household contacts, and identify potential risk factors. this investigation occurred in the emirate of abu dhabi, which occupies >80% of the uae's total area [16] and is comprised of 3 regions: abu dhabi (capital city), al ain region, and al dhafra. the emirate of abu dhabi has a population of 2.8 million (2015 estimate) [17] . the al ain region borders oman and saudi arabia and houses the second largest city in the emirate, al ain city. while al ain city is an oasis, the rest of the region primarily consists of desert and mountains. the al dhafra region is mainly desert and rural with approximately 285 000 residents and a population density of 8 residents/km 2 [18] . all laboratory-confirmed mers-cov cases (n = 65) in the emirate of abu dhabi diagnosed between 1 january 2013 and 9 may 2014 and their household contacts (n = 452) were eligible for the investigation. these cases were a convenience sample during the ongoing mers-cov outbreak. two of the 431 (0.5%) household contacts tested for mers-cov during initial contact investigations were pcr positive and eligible to be enrolled as cases for our investigation (figure 1 ). the enrolled case was a healthcare worker who might have been exposed by another coworker, who also lived in the case's household; therefore, the enrolled case was a result of either household or healthcare transmission prior to this investigation's initiation. the case not enrolled in this investigation was exposed in the household. household contacts were defined as any person who stayed at least 1 night at the same location as the case patient during the 14 days prior to the case patient's symptom onset or the date of first positive specimen if the case patient was asymptomatic. excluded cases included palace workers and other high-level officials; their associated household contacts were also excluded. for each mers-cov case identified in the investigation, clinical information, including symptoms, was collected using the international severe acute respiratory and emerging infection consortium form, which was filled out in real time by healthcare providers and subsequently verified by retrospective chart review. in abu dhabi during this time period, all individuals who tested positive for mers-cov were admitted to a healthcare facility for observation and infection control regardless of symptom status. the same definitions for case severity were used as in al hosani et al [5] including the following: asymptomatic cases reported no symptoms at the time of a positive test as recorded by a healthcare provider in the medical chart; mildly symptomatic cases reported symptoms, such as pharyngitis, rhinorrhea, or cough, and did not require oxygen during their hospitalization; and severely symptomatic cases required supplemental oxygenation during their hospitalization, ranging from nasal cannula to mechanical ventilation. using data collected from doh's surveillance of mers-cov cases, households with mers-cov case patients were approached. household contacts who were eligible for the investigation included those that had been identified through contact investigations associated with the case patient performed by doh officials within 24 hours' notification. three attempts were made to contact each household. if no response was received after 3 attempts, the household was not enrolled. households that agreed to be enrolled were given an appointment at the local disease prevention and screening center for questionnaire administration and serum collection. questionnaires were administered in english, arabic, or, if an interpreter was available, the participant's native language. data collected included demographics; residence/household description; exposure history to other mers-cov cases, healthcare settings, and animals; travel history; and medical history, including any long-term effects reported by case patients. for deceased case patients, a proxy completed the case patient questionnaire using recall. the real-time reverse-transcription pcr (rrt-pcr) results were obtained from the doh surveillance data. upper (ie, nasopharyngeal, oropharyngeal) and lower respiratory tract specimens (ie, sputum, bronchoalveolar lavage fluid, tracheal aspirates) were analyzed using rrt-pcr in the sheikh khalifa medical center laboratory. additional laboratory result verifications were performed in a random sample of 23 specimens using nucleocapsid-based rrt-pcr [5] . serum samples were inactivated using 2 × 10 6 rads gamma irradiation and stored at ≤ -70°c until use. screening of serum specimens by mers-cov nucleocapsid enzyme-linked immunosorbent assay (elisa) was performed at the sheik khalifa medical city in abu dhabi, uae and the centers for disease control and prevention (cdc), atlanta, georgia. titers of ≥1:400 were reported as positive. recombinant full length mers-cov nucleocapsid protein indirect elisa was used to screen serum specimens as described by al-abdallat et al [19] . serum samples were tested for the presence of neutralizing antibodies to mers-cov using a microneutralization assay (mnt) [19] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell lysis in at least 1 of the 3 triplicate wells. positive and negative controls were included for each mnt performed and included back-titration and mock-infected cells. titers of ≥1:20 were reported as positive. all work with live mers-cov was done in biosafety level 3 containment at the cdc. immunofluorescence assays (ifas) were performed by screening sera at a dilution of 1:50 and 1:100 on paraformaldehyde-fixed, acetone-methanol permeabilized mers-cov (strain mers-cov hu/england-n1/2012) infected or uninfected control vero cells. antihuman immunoglobulin g, m, and a fluorescein isothiocyanate conjugate was used to detect anti-mers-cov antibodies in human serum, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole to allow identification of individual mers-cov-infected cells. fluorescence was detected using a zeiss axioimager fluorescence microscope. the positive control for the assay is a serum sample from a patient infected with mers-cov hu/ england-n1/2012. a positive result was scored when these 3 conditions were met: cells were evenly stained (instead of punctate staining); fluorescence intensity was higher than that of the negative controls; and signal intensity declined with serial dilution. a minimum of 2 negative controls were included with each ifa. approximately 10% of specimens negative by nucleocapsid elisa were screened by both ifa and mnt to confirm the negative result. mers-cov antibody positivity was defined as one of the following: (1) 2 of 3 tests (ie, mers-cov nucleocapsid elisa, mers-cov mnt, and ifa) were positive; or (2) mers co-v mnt was the only positive test. household survey data were entered into electronic forms in epi info 7 version 7.1 (cdc). quality control and assurance were performed through epi info 7 intelligent codes programmed into the forms. household survey data were merged with the laboratory results, and descriptive analysis was completed. differences in proportions were compared using the mantel-haenszel χ 2 test, while differences in continuous variables were compared using the student t test. p < .05 was considered statistically significant. data analysis of the merged dataset was conducted with sas version 9.3 software (sas institute, cary, north carolina). following local customs, informed consent was obtained from the head of the household, who provided consent for all members of a household; however, each individual was still able to decline participation. this investigation was determined by doh and cdc to be part of a public health response, not research, and therefore not subject to institutional review board review. thirty-four case patients' households were included (supplementary table 1 ). household residences ranged in size from 7 m 2 to 1100 m 2 (interquartile range [iqr], 70-200 m 2 ). a median of 4 individuals (range, 1-30) lived in the households 14 days prior to the diagnosis of a mers-cov household case patient. more than half of mers-cov case patients shared a bathroom with others in the household. all households reported having air conditioning. thirty-four cases of 65 (52%) and 124 household contacts of 452 (27%) participated (table 1) . females comprised a higher proportion of case patients compared with household contacts (70.6% vs 53.2%), and case patients were older compared with household contacts (median, 42 years vs 31 years). most case patients and contacts were from the al ain region of the abu dhabi emirate. seventy-one percent (n = 24) of case patients reported working in a healthcare setting 14 days prior to diagnosis, with nurses being most represented (24%, n = 8) ( table 1) ; only 24% (n = 30) of household contacts worked in a healthcare setting 14 days prior to a case patient's diagnosis. compared with household contacts, case patients less frequently reported visiting or owning a farm (12% vs 14%), but reported camel exposure more frequently (12% vs 7%). household contacts reported the following frequent exposures to mers-cov case patients: hugging (54%, n = 67), using the same bathroom (51%, n = 63), sharing meals (49%, n = 61), and kissing or nose-kissing (ie, rubbing tips of noses against one another) (48%, n = 60). case patients reported a higher proportion of underlying medical conditions than household contacts, including diabetes (18% vs 5%, p = .01), hypertension (27% vs 7%, p < .001), kidney failure (6% vs 2%, p = .03), and heart failure (6% vs 0%, p < .01) ( table 1 ). case patients also reported taking medications for any illness more frequently than contacts (38% vs 17%). three case patients (9%) reported limitation to activities due to mers-cov with a median duration of 5 days (iqr, 4-24 days) ( table 1) . normal activities were resumed at a median of 5 days (iqr, 3-7 days). of 34 case patients, 17 (50%) reported being asymptomatic, 14 (41%) reported being mildly symptomatic, and 3 (9%) were severely symptomatic. age and proportion having underlying medical conditions increased with symptom severity ( table 2) . symptom duration did not have any noticeable trend with symptom severity (data not shown). all severe case patients were treated in the intensive care unit, as well as 1 asymptomatic case, who had underlying diabetes, hypertension, and kidney disease. the median days hospitalized increased with symptom severity ( table 2) . sera were obtained from 24 of 34 (71%) case patients and 105 of 124 (85%) household contacts. among the 24 case patients with available sera (table 3) among the 13 case patients with detectable antibodies against mers-cov, all of them were aged <60 years, with a median age of 43 years, compared to a median of 32 years for case patients without detectable antibodies (table 4 , p = .04). number of days of pcr positivity was notably higher among those who had detectable antibodies compared to those who did not (median, 15 days vs 2 days, p = .01). we describe the results of follow-up of 34 mers-cov case patients and 124 of their household contacts from the emirate of abu dhabi during 2013-2014. notably, serologic testing did not find any evidence of mers-cov transmission in the households of mers-cov case patients in our investigation, suggesting that viral transmission from asymptomatic or mildly symptomatic individuals to household contacts does not readily occur. sera were tested with a combination of 3 different laboratory assays (nucleocapsid elisa, ifa, and mnt); we feel confident that individuals identified as "negative" did not seroconvert. although there was clear evidence of household transmission in 1 household not enrolled in this investigation, our investigation's results did not show evidence of additional household transmission. overall, our findings support current recommendations that home isolation may be appropriate for asymptomatic cases and close contacts who are ill and do not require hospitalization in consultation with local public health departments [20, 21] . because this investigation occurred during may-june 2014, many case patients were recruited from the april 2014 healthcare-associated outbreak at an al ain region hospital [22] . a kingdom of saudi arabia study found that while healthcare personnel were at high risk for infection, most illness was relatively mild and could be unrecognized, highlighting potential undetected transmission of the virus to others [23] . in our investigation, case patients tended to be younger (30-59 years) , and most reported working in a healthcare setting 14 days prior to their diagnosis where they were exposed to a similar to previous studies, case patients with severe disease had higher frequency of comorbid conditions and required intensive care, including intubation [24] [25] [26] . in a recent investigation from south korea, patients with a higher host infectivity, which included evaluation of pcr cycle threshold values, along with higher numbers of contacts, were more likely to transmit mers-cov [27] . it is likely that most of the primary case patients in this investigation had lower host infectivity. while our investigation found that some asymptomatic or mildly symptomatic case patients had detectable antibodies, we did not find any detectable antibodies in 11 asymptomatic and mildly symptomatic case patients. other studies also did not find detectable antibodies in some asymptomatic and mildly ill cases [6, 28] . if seroconversion is to occur in case patients, studies have demonstrated that this usually occurs within the first month of illness [28] [29] [30] . for the majority of case patients with detectable antibodies, we found persistence of antibody response for several months after the initial diagnosis, even close to a year. additionally, these case patients had a longer duration of mers-cov pcr positivity than those who did not have detectable antibodies, indicating a potential relationship between longer viral shedding and seroconversion. previous studies have demonstrated that asymptomatic and mildly symptomatic case patients can test pcr positive >2 weeks from lower respiratory tract specimens [5, 31] . our investigation's serology results do not provide additional evidence of transmission to household contacts, though there is evidence from other settings to suggest limited household transmission [11] . also, very low rates of household transmission have been reported during hospital-based outbreaks [19, 32] . more robust transmission studies involving larger numbers of case patients representing a range of clinical and demographic characteristics and their contacts are needed to further investigate risk exposures. there are several limitations to this investigation. first, serum samples were collected at varying intervals after illness onset for each case patient, potentially affecting serology results. the duration of antibody response is unknown. second, recall bias might have led to the misclassification of symptom severity among household contacts; however, for case patients, to minimize this bias, we relied upon retrospective medical chart review, though this also might not be as complete since it depended on the initial healthcare provider's history and physical. third, these case patients were immediately isolated in hospitals after pcrpositive results were discovered. the removal from the household setting might have reduced exposure to household contacts although the case patients were residing with household contacts at the time of the contact investigations. last, because our investigation did not detect household transmission, we cannot comment on any behaviors or exposures that would increase risk among household contacts of case patients. in summary, we did not document additional household transmission in this investigation that included a preponderance of asymptomatic and mildly symptomatic confirmed mers-cov case patients. our investigation findings support the recommendation to consider home isolation for asymptomatic and mildly ill cases that do not require hospitalization while using proper precautions, including face masks, frequent hand washing, and minimizing exposure to the case patient in the household [20, 21, 33] . while no vaccines or antivirals against mers-cov are currently available, reducing transmission through effective infection control management remains a major priority. understanding transmission risk for different mers-cov-infected patients who live in different settings will be important data that must be factored into prevention strategies. further studies on human-to-human transmission in different settings should be conducted to inform mers-cov prevention and control guidelines. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory 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infection: a single-center experience in saudi arabia middle east respiratory syndrome risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea serologic responses of 42 mers-coronavirusinfected patients according to the disease severity viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker health care worker contact with mers patient, saudi arabia interim guidance for healthcare professionals acknowledgments. the authors gratefully thank the participants; the teams from the disease prevention and screening center-al ain, the disease prevention and screening center-abu dhabi, ghayathi hospital, and silla hospital that supported this investigation; and suvang trivedi and seyhan boyoglu-barnum at the centers for disease control and prevention (cdc) for technical support.disclaimer. the conclusions, findings, and opinions expressed herein are those of the authors and do not necessarily reflect the official position of the us department of health and human services, the us public health service, the cdc, or the authors' affiliated institutions.financial support. this work was supported by the cdc. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-330343-p7a8chn4 authors: kelly-cirino, cassandra; mazzola, laura t; chua, arlene; oxenford, christopher j; van kerkhove, maria d title: an updated roadmap for mers-cov research and product development: focus on diagnostics date: 2019-02-01 journal: bmj glob health doi: 10.1136/bmjgh-2018-001105 sha: doc_id: 330343 cord_uid: p7a8chn4 diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. ► the middle east respiratory syndrome-coronavirus is a high-priority pathogen identified by the who r&d blueprint because of its high fatality rate, large geographical range of the dromedary camel reservoir and lack of medical interventions. ► accurate and accessible diagnostic tests are essential to outbreak containment and case management, as well as surveillance in both humans and animals, but available diagnostic tests are limited by inconsistent quality assessment, specimen acquisition issues and infrastructure requirements. ► diagnostic research and development (r&d) needs to include point-of-care testing options, syndromic panels for differential diagnosis, a greater understanding of viral and antibody kinetics, improved access to clinical specimens, and establishment of international reference standards. diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. the middle east respiratory syndrome-coronavirus (mers-cov) is an emerging virus associated with severe respiratory illness, first detected in 2012 in saudi arabia. 1 5 provides an overview to the current status of mers-cov diagnostics, including feedback from subject matter expert and developer interviews on the common challenges with test development and implementation, and identifies gaps for further research and development (r&d). mers-cov is a zoonotic virus, and dromedary camels (camelus dromedarius) are the reservoir host and the source of zoonotic transmission to humans. [6] [7] [8] dromedaries appear to be only mildly symptomatic following infection and present a significant reservoir risk for spillover events. 2 6 9 mers-cov rna has been detected in dromedary camels in a number of countries, including egypt, oman, qatar and saudi arabia, with evidence suggesting that mers-cov is also widespread in the middle east, africa and south asia. 5 8 10-35 infection in camels is notifiable to the oie. 36 individuals with close and frequent contact with dromedaries are at a higher risk for mers-cov infection than the general population. 37 38 clinical indications and management coronaviruses are a family of viruses that can cause diseases in humans, ranging from the common cold to severe acute respiratory syndrome (sars). the clinical spectrum of mers ranges from no symptoms (or asymptomatic infection), mild symptoms including fever, cough, gastrointestinal illness and shortness of breath, to severe disease including pneumonia, acute respiratory distress syndrome and death. 2 39 severe cases of mers can result in respiratory failure, requiring mechanical ventilation and support in intensive care. risk factors for severe disease include a weakened immune system, older age (>60 years), and comorbidities such as diabetes, cancer, renal disease and chronic lung disease. 40 41 human-tohuman transmission spreads through close and unprotected human contact, and more than half of reported mers cases have occurred through nosocomial transmission. [42] [43] [44] [45] to prevent nosocomial infections, who and others recommend using standard infection and prevention control measures when caring for patients. [46] [47] [48] who also recommends that contact tracing of all symptomatic and asymptomatic close contacts of the primary patient should be conducted routinely. 49 the molecular epidemiology for mers-cov has not changed significantly since the initial human cases were detected in 2012. the current virus remains 99% identical to the sequences seen in the first human cases from 2012 as well as archived camel sera from 1983, with no increase in pathogenicity observed in the animal host. [50] [51] [52] as genetic mutations could impact detection, bmj global health immunotherapy and vaccine development efforts, 53 sequencing of mers-cov strains from camels and humans (after a zoonotic spillover) is important and is regularly being conducted in affected member states (who, personal communication, 2018). there are currently no prophylactic or therapeutic interventions of proven efficacy for any coronavirus infections. without a specific therapy for mers, treatment is supportive. 5 54 55 effective mers therapeutics are still in the early stages of research and evaluation. several broad-spectrum antiviral agents including nitazoxanide, 56 viral methyltransferase inhibition 57 and nucleotide prodrugs 58 have shown in vitro activity against mers-cov. early results for novel mers-specific therapeutics that inhibit viral replication or have specific neutralising activity are promising. 47 59 60 the who r&d blueprint for mers has called for three types of vaccines: (1) dromedary camel vaccine to prevent zoonotic transmission, (2) human vaccine for long-term protection of persons at high exposure risk and (3) human vaccine for reactive use in outbreak settings. 55 61 mers-cov vaccines are in the early stages of development, 55 62 63 with one candidate vaccine in phase i clinical trials (nct02670187). 64 neutralising monoclonal antibodies have been designed to target the mers-cov spike protein, 53 65 with chadox1 and modified vaccinia ankara vectors also strong vaccine candidates, 60 66 but none have yet advanced to clinical trials. to accelerate the process, the coalition for epidemic preparedness innovation has recently launched a call for proposals for the development of a human mers-cov vaccine in order to engage with developers interested in supporting these efforts. 67 the who laboratory guidelines recommend nucleic acid amplification tests (naat) for diagnosis, using serology for diagnosis only when naat is not available. 68 in suspected patients, a single negative test result does not exclude diagnosis. repeat sequential sampling and testing is strongly recommended. the kinetics of mers-cov infection has been shown to vary widely across cases, 40 69-72 prompting a more detailed investigation of viral and antibody dynamics across the broad range of sample types, disease states and host factors. 73 74 the best naat test sensitivity is achieved using specimens from the lower respiratory tract (sputum, tracheal aspirates or bronchoalveolar lavage), where mers-cov replication occurs at higher and more prolonged levels of mers-cov rna, typically between 10 6 and 10 10 copies/ml. 72 75 mers-cov viral load is generally higher for severe cases, with more prolonged viral shedding than mild cases. viral load concentrations, which may be undetectable at early-stage infection, generally peak in the second week after symptom onset, and then drop to undetectable in survivors by the fourth week from onset. upper respiratory tract specimens (nasopharyngeal or oropharyngeal swabs) may also be used, but demonstrate 100×-1000× lower viral load and can test negative for mild cases. 76 77 if possible, both upper and lower respiratory tract sampling are advised. specimens outside the respiratory tract are not recommended for diagnosis, as they can test negative in both severe and mild presentation. viral rna has been detected in stool samples (10 4 copies/ml), serum samples (10 3 copies/ml) and urine (10 2 copies/ml), more likely an indicator of severity as it typically precedes a poor clinical outcome. 71 76 78 serological diagnosis can be made using paired samples, more often used for research rather than diagnostic purposes, preferably with the initial sample collected in the first week of illness and the second collected 3-4 weeks later. if only a single serum sample can be collected, this should occur at least 3-4 weeks after onset of symptoms for determination of a probable case. table 1 presents an overview of the implementation requirements for mers-cov diagnostics (detailed commercial product information is presented in online supplementary tables s1 and s2). molecular diagnostics such as naat (eg, pcr) typically require sophisticated laboratory infrastructure including biosafety cabinets, 79 while most serological tests (elisa, indirect immunofluorescence test (iift)) can be run on the benchtop in a more modest laboratory environment, depending on sample preparation precautions. 80 81 point-of-care (poc) tests are designed to be used outside of a traditional laboratory; near-poc tests are defined for rapid use in a laboratory near the patient, but are more automated and easy to use than the traditional laboratory test. 72 75 poc tests such as low-complexity rapid diagnostic tests (rdts) can be used at the bedside, typically with non-invasive samples after minimal training. inhouse tests are described in sections below; commercial sources are listed in online supplementary tables s1 and s2. naats are currently the standard for mers-cov diagnosis, as these tests (typically reverse transcriptase pcr (rt-pcr)) have the highest sensitivity at the earliest time point during the acute phase of infection. following the who guidelines, two different targets on the mers-cov need to be detected by rt-pcr to confirm a case. mers-cov assays to detect the upstream envelope gene (upe) followed by confirmation of open reading frame 1a (orf1a), 1b (orf1b) genes or nucleocapsid (n) genes for confirmation have been developed. 55 82 most commercial pcr tests perform parallel screening for the upe gene with confirmation by the orf1a, orf1b or n genes (most commonly upe + orf1a). initial naat tests for mers-cov were developed as inhouse tests, following the first detection of mers-cov in the middle east. [83] [84] [85] [86] inhouse tests are not necessarily subject to quality control or regulation, and may not be rigorously validated; in some cases, inhouse tests are eventually developed into commercial products through collaboration and licensing efforts. 50 83 84 87-89 commercial assays may undergo an international and/or incountry regulatory process; once on the market they can be independently evaluated for sensitivity, specificity and limit of detection. 78 90 as of 2018, there are several commercial naat tests available for mers-cov, including duplex and multiplex panels (see online supplementary table s1). serology is not widely performed for diagnosing acute mers-cov infection; however, it has been a useful tool bmj global health to determine the extent of infection around clusters and in seroepidemiological studies in animals and humans. seroconversion typically occurs during the second and third week after symptom onset; data suggest that low antibody titre in the second week or delayed seroconversion is more closely associated with mortality than high viral load. 71 74 mers-cov seroconversion may not be observed for some patients, notably with mild or asymptomatic infection, and can show cross-reactivity with antibodies to other coronaviruses. 42 69 serological methods for the detection of antibodies against mers-cov include elisa, iift and neutralisation tests. mers-cov serological assays can employ commercial reagents or proprietary monoclonal antibodies as capture agents. 87 91 92 many mers-cov elisa tests are labelled for research use only, with little or no clinical validation data available. similar to the elisa, iift is used when it is difficult to evaluate specific antigens individually by enzyme immunoassays or there is a preference for broader analysis of an immobilised specimen. iift microscopy assay can probe the entire antigen spectrum of the specimen, and is often designed for simultaneous detection of antibodies against biochemically distinct antigens. neutralisation is a method for detecting anti-mers-cov antibody activity via inhibition of infection or replication, 69 93 performed as plaque reduction neutralisation, microneutralisation (mn) and pseudoparticle neutralisation (ppnt). mn is labour-intensive and slow, requiring at least 3-5 days for results; neutralisation techniques other than ppnt require biosafety level 3 containment as they involve live virus cultures. 94 rdts can leverage the same antibody/antigen capture agents as elisa but in a lateral flow strip cartridge. 95 this enables a fast 10-30 min time to result, but with a 100-fold lower detection sensitivity than elisa. 91 92 follow-up confirmatory testing is therefore required. rdts are typically paired with minimally invasive specimen collection (blood, oral fluid, nasal swabs) so that they can be used with minimal training outside of laboratory settings. early prototypes for mers-cov rdts have been developed, 87 92 96 with commercial rdts for detection of mers-cov in camels and humans available (online supplementary table s2). the human mers-cov rdt does not appear to be widely used, perhaps due to the more invasive processing required for lower respiratory specimens, as well as sensitivity issues for upper respiratory specimens. the camel mers-cov rdt is used with upper respiratory specimens; however, test sensitivity varies depending on specimen sampling and infection kinetics. 97 multiplex panels at the early stages, the symptoms of mers-cov infection can mimic diseases such as influenza, pneumonia, sars and other respiratory infections. a syndromic approach involves testing for pathogens based on a syndrome such as fever or acute respiratory distress; a shift from individual tests to multiplex panels can quickly identify or eliminate likely pathogens from a single specimen. for analysis of circulating reservoirs, multiplex microbead-based immunoassays have been used to detect igg antibodies for multiple pathogens. 98 99 multiplex, syndromic panels that include mers-cov have been demonstrated using pcr-based panels including mers-cov, showing similar limits of detection to single assays. 89 100 101 commercial respiratory panel tests including mers-cov have also recently been developed (see online supplementary table s1). there is a need for international consensus and adoption of minimum standards for tests used in diagnosis, surveillance and research, following who's recommended algorithm for human cases 82 and oie recommendation for animal health. 36 harmonisation of the testing process can be achieved by building consensus and capacity across international and incountry laboratories. in order to enable and sustain the capacity for a rapid outbreak response, laboratories must have access to high-quality reagents and instrumentation, along with technical support and cold-chain transport when necessary. in addition, international reference panels would achieve a more standardised training for external quality assessment (eqa) and quality control. building on mandatory case reporting, 102 an international mers-cov data sharing platform that includes case exposure history and sequence data would greatly facilitate the knowledge base across the mers-cov community. [103] [104] [105] [106] clinical validation understanding mers-cov viral dynamics across a broad range of specimen types is critical to establishing the limits of detection and timing of diagnostics in order to make the greatest impact for diagnosis, case management and surveillance. ensuring a test has appropriate sensitivity and specificity is a major challenge in the development of diagnostics for novel and rare pathogens, as there is often a very limited supply of well-characterised clinical material. especially during the early stage of an outbreak, clinical evaluation must often be performed in the affected countries by laboratories working closely with the ministries of health. typically only a small number of patient specimens are shared outside of the affected countries due to strict import and export regulations, particularly for 'dual-use' pathogens. 107 108 specifically, the provisions of the nagoya protocol have significant impact on the access to genetic materials for both commercial and non-commercial applications. 109 110 in particular, the development and validation process for new diagnostics could be accelerated if well-characterised specimens and reference standards could be more easily obtained. eqa can be useful for evaluation of test performance, as shown with evaluations of both inhouse and commercial assays for mers-cov, [111] [112] [113] and bmj global health more recently a global proficiency testing programme used to assess laboratory detection of mers-cov. 114 even after validation, a substantial amount of reference material is required for quality control; often manufacturers must develop their own calibration standards to maintain supply and to control lot-to-lot variability. international reference standards and qualified specimen panels can accelerate the development and validation of diagnostic tests. in particular, the who international biological reference preparations (as provided by member states) serve as reference sources for ensuring the reliability of in vitro biological diagnostic procedures used for diagnosis of diseases and treatment monitoring, including mers-cov. several international institutes also provide specimens for validation; these groups typically have a defined pathogen/disease focus with a corresponding archive of biological reference materials; however, the supplies may be limited (see online supplementary material 1). currently, mers-cov diagnosis by pcr requires a laboratory with sophisticated facilities and biosafety cabinets. the turnaround time to receive a test result can take days to weeks, depending on laboratory proximity, sample transport options and laboratory processing capacity, 72 75 and infrastructure requirements place most pcr systems in reference laboratories, which may not be ideal for diseases like mers-cov that recommend immediate isolation for infections detected across a variety of settings. 81 115 116 a more nimble approach is needed for mers-cov case detection and triage, 92 117 and at border crossings for animal surveillance, quarantine and targeted vaccination. 11 21 87 118 the fao-oie-who mers technical working group has given a clear call for the development of an rdt to improve identification and isolation of primary human cases in healthcare facilities. 5 serological rdts are ideal for low infrastructure settings such as a primary health clinic, home or field testing. however, specimen collection remains a key challenge for mers-cov, as the recommended lower respiratory specimens are difficult to obtain outside of a hospital setting. upper respiratory specimens such as nasal swabs are easy to obtain and work well in conjunction with rdts for camels, but these specimens generally have low virus titre in humans, thus limiting current use of rdts to animal testing. 87 92 96 improvement of the current rdt detection chemistry, if feasible, may support the future use of these tests in humans, at least for rapid triage in highly infectious cases. poc and near-poc microfluidic platforms enable a more flexible, but still highly sensitive approach for near-patient naat testing in decentralised settings. near-poc naat platforms are compact and self-contained, with automated sample preparation for processing in minimal laboratory settings, which most healthcare workers can be trained to operate within a day. [119] [120] [121] recent publications describe mers-cov assays designed for poc pcr, 89 loop-mediated isothermal amplification assay 122 and paper-based sensor detection 123 ; however, no mers-cov assays are currently available for the existing near-poc platforms. given that pcr is now the standard for mers-cov diagnosis, it would be highly desirable to have an automated, self-contained naat assay that can be readily deployed in a field or clinic setting. syndromic testing can be valuable during the early stages of an outbreak, in order to distinguish mers-cov from other respiratory infections or identify cases of coinfection. 100 124 a syndromic panel could be effective in expediting pathogen and outbreak identification, especially with technologies that can screen for multiple pathogens simultaneously. 125 using the panel approach, a definitive diagnosis could enable timely decisions about triage, treatment, infection control and contact tracing. 126 while the per-test cost rises with test complexity, including additional reagents and more sophisticated instrumentation, a rapid and efficient diagnosis scheme can impact intervention and infection control and can be cost-saving overall. 127 128 as respiratory diseases are both regional and seasonal, 129-131 region-specific panels may be more cost-effective. 132 multiplex panels offer the alternative for a 'bundled' testing paradigm; however, if not routinely used (if the market is small), then developers may be reluctant to support the test for diagnostic use, which requires additional investment for validation and regulation. surveillance can be an effective method to identify the initial stages of outbreak, but it requires routine and effective sampling. the impact of surveillance testing depends on the test sensitivity and specificity, sampling rates, kinetics of the disease, and whether the target is animal or human populations. most surveillance sampling is performed in the field, either through population-based or 'hot spot' sampling. for mers-cov, it may be difficult and expensive to implement routine surveillance in dromedary camel stock, as they represent a significantly large reservoir but may suffer only mild effects from mers-cov infection, if any. the ideal surveillance tool would be a highly sensitive and field-appropriate screening test. per-test cost is also an important factor along with ease of implementation. this review has identified diagnostics currently available for mers-cov and highlighted ongoing challenges caused by critical gaps in diagnostics to support outbreak management. rdts offer the potential for rapid poc screening for mers-cov; however, there are practical limits to implementing lower respiratory sample acquisition outside of a hospital setting, limiting feasibility. poc or near-poc naat platforms provide an opportunity for implementation of automated, self-contained bmj global health testing in hospitals and clinics with limited training in endemic-prone areas. expansion of test menu options for existing poc or near-poc naat platforms will strengthen incountry response capacity to endemic diseases and simultaneously ensure countries are prepared for future pandemics. syndromic multiplex panels may expedite differential diagnosis of mers-cov from other endemic respiratory diseases, but further analysis is needed to inform implementation and cost-effectiveness in the context of regional and seasonal detection. there is also a need for more sensitive serological assays with lower cost and minimum cross-reactivity that can be used as surveillance tools. a more detailed understanding of mers-cov viral and antibody kinetics is needed across the broad range of sample types in order to optimise the use of existing assays and to address ongoing technical challenges in the detection of mild and asymptomatic infections. surveillance continues to be important for the detection of mers-cov spillover events; however, questions remain 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diseases in the asia pacific region point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens cost analysis of multiplex pcr testing for diagnosing respiratory virus infections impact of a rapid respiratory panel test on patient outcomes pcr for detection of respiratory viruses: seasonal variations of virus infections global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study prevalence and seasonal distribution of respiratory viruses during the 2014 -2015 season in istanbul development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay acknowledgements we gratefully acknowledge input to the roadmap from all those who attended the fao-oie-who global technical meeting on mers-cov in september 2017. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organisations with which they are affiliated. editorial assistance for later drafts was provided by rachel key: cord-331228-wbd0s4fo authors: shehata, mahmoud m.; gomaa, mokhtar r.; ali, mohamed a.; kayali, ghazi title: middle east respiratory syndrome coronavirus: a comprehensive review date: 2016-01-20 journal: front med doi: 10.1007/s11684-016-0430-6 sha: doc_id: 331228 cord_uid: wbd0s4fo the middle east respiratory syndrome coronavirus was first identified in 2012 and has since then remained uncontrolled. cases have been mostly reported in the middle east, however travel-associated cases and outbreaks have also occurred. nosocomial and zoonotic transmission of the virus appear to be the most important routes. the infection is severe and highly fatal thus necessitating rapid and efficacious interventions. here, we performed a comprehensive review of published literature and summarized the epidemiology of the virus. in addition, we summarized the virological aspects of the infection and reviewed the animal models used as well as vaccination and antiviral tested against it. coronaviruses (cov) became known to cause human disease in the twentieth century. hcov-229e and hcov-oc43 were discovered in the 1960s and shown to cause respiratory infections in humans [1, 2] . with the emergence of sars-cov in 2003 [3] , two other human coronaviruses were discovered, hcov nl63, hcov hku1 [4] . in 2012, a new type of coronavirus was detected as the cause of severe respiratory illness in humans. the first case was a 60-year-old male from saudi arabia admitted to hospital with acute respiratory illness leading to pneumonia and acute renal failure. the virus initially named as human corona virus-emc [5] , is currently known as the middle east respiratory syndrome coronavirus (mers-cov) [6] . classification and nomenclature of mers-cov phylogenetically, mers-cov is a lineage c β coronavirus (β-cov) and is closely related to bat coronaviruses hku4 and hku5. the rooted phylogenetic analysis showed that mers-cov had an amino acid sequence identity less than 90% to all other known covs [7] . the virus initially named by many different working groups as novel coronavirus, human coronavirus emc, human b coronavirus 2c emc, human b coronavirus 2c england-qatar, human b coronavirus 2c jordan-n3, and b coronavirus england 1, which represented the places where the first complete viral genome was sequenced (erasmus medical center, rotterdam, the netherlands) or where the first laboratory-confirmed cases were identified or managed (jordan, qatar, and england) was later named as mers-cov by the coronaviruses study groups of ictv [5, 6, 8] . mers-cov is an enveloped virus with a positive sense rna genome. coronavirus genomes range between 25 to 32 kb in size. the complete sequence of hcov-emc-2012 resulted in 30 119 nucleotides sequence [7] . coronavirus genomes are polycistronic with large replicase open reading frames orf1a and orf1b which are subsequently cleaved into 15 or 16 nonstructural proteins (nsps). the region downstream of orf1b encode smaller genes including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural protein [9] [10] [11] . the functional receptor for mers-cov is the dipeptidyl peptidase 4 (dpp4) which is present on human nonciliated bronchial epithelial cells surfaces [12] . the dpp4 protein displays high amino acid sequence conservation across different species, including the sequence that was obtained from bat cells. cell lines susceptibility studies showed that mers-cov infected several human cell lines, including histiocytes as well as respiratory, kidney, intestinal, and liver cells [13] . the range of tissue tropism in vitro was broader than that for any other known human coronavirus [14] . mers-cov can also infect nonhuman primate, porcine, bat, civet, rabbit, and horse cell lines all possessing the dpp4 receptor [15] . the replication cycle of mers-cov consists of numerous steps as illustrated by lu et al. [30] . the mers-cov s protein is a class i fusion protein composed of two subunits: the amino n-terminal receptor binding s1 and carboxyl c-terminal membrane fusion s2 subunits. the s1/s2 junction is a protease cleavage site which is responsible for membrane fusion activation, virus entry, and syncytium formation. the s1 c domain contains the receptor binding domain (rbd), and an n domain [13] . neutralizing monoclonal antibodies against the rbd may inhibit virus entry into cells and receptor-dependent syncytium formation in cell culture, hence vaccines containing the rbd induced high levels of neutralizing antibodies in mice and rabbits [16] [17] [18] . dpp4 is the cell key functional receptor for the mers-cov s protein [19] . mers-cov is the first cov that has been identified to use dpp4 as a receptor [19, 20] . dpp4 has important roles in glucose metabolism, t cell activation, chemotaxis modulation, cell adhesion, and apoptosis [19, 21] . the s2 subunit contains five domains: a fusion peptide, the heptad repeat 1 (hr1) and hr2 domains, a transmembrane domain, and a cytoplasmic domain, which form the stalk region of s protein that facilitates fusion of the viral and cell membranes [22, 23] . the binding of the s1 subunit to the cellular receptor triggers conformational changes in the s2 subunit, which inserts its fusion peptide into the target cell membrane to form a six-helix bundle fusion core between the hr1 and hr2 domains that approximates the viral and cell membranes for fusion. mers-cov utilizes many pathways for membrane fusion depending on available host proteases, such as transmembrane protease serine protease 2 (tmprss2), trypsin, chymotrypsin, elastase, thermolysin, endoproteinase lys-c, and human airway trypsin-like protease. proteases cleave the s protein into the s1 and s2 subunits to activate the mers-cov s protein for endosome-independent host cell entry at the plasma membrane [24] [25] [26] . in addition to the pervious fusion proteases furin has been identified recently to play an essential role in the mers-cov s protein cleavage activation into their biologically active forms [27, 28] . after cell entry, the virion particle disassembles to release the nucleocapsid and viral rna into the cytoplasm for expression of viral polyproteins pp1a and pp1ab. doublemembrane vesicles and convoluted membranes are formed by the attachment of the hydrophobic domains of the mers-cov replication machinery to the limiting membrane of auto-phagosomes [29] . the viral polyproteins pp1a and pp1ab are cleaved by papain-like protease and 3c-like protease into nsp1 to nsp16 [7, 30, 31] . these nonstructural proteins form the replication-transcription complex, which regulates transcription and viral protein expression [29] . after the production of abundant viral rna and structural and accessory proteins, the n protein binds to the genomic rna in the cytoplasm to form the helical nucleocapsid (viral core). the viral core is enveloped by budding through intracellular membranes between the endoplasmic reticulum and golgi apparatus [32] . the s, e, and m proteins are transported to the budding virion, where the nucleocapsid probably interacts with m protein to generate the basic structure and complexes with the s and e proteins to induce viral budding and release from the golgi apparatus [33] . mers-cov replication cycle is completed by releasing the progeny virions through the cell membrane via exocytosis pathway. mice mers-cov strain hcov-emc/2012 was inoculated to three different mouse strains (immunocompetent balb/c mice, 129s6/svev and innate immune-deficient 129/ stat1 -/mice) intranasally. no significant weight loss was observed and infectious virus could not be detected in the lungs. only moderate pathological lesions were observed in the lungs. hence no viral replication was observed in these strains of mice [34] . zhao et al. developed a mouse model transduced with a recombinant adenovirus vector expressing hdpp4 (ad5-hdpp4) in lung tissue. inoculation of mers-cov in these mice resulted in mers-cov replication but without mortality. young mice cleared from mers-cov in 6-8 days and old mice in 10-14 days. perivascular and peribronchial lymphoid infiltration was observed, with progression to an interstitial pneumonia postinfection [35] . in another study, transgenic mice expressing hdpp4 were susceptible to mers-cov infection. infectious virus was isolated from lung and brain tissue and weight loss was observed [36] . pascal et al. developed humanized transgenic mouse. no mortality or clinical signs was observed but interstitial pneumonia and significant lung disease were observed histopathologically, suggesting that humanized dpp4 mouse is a model for mers-cov infection in which pathological changes resembles mers-cov infection in humans [37, 38] . the rhesus macaque was the first animal model used for mers-cov infection as it possessed dpp4 receptor [38, 39] . in infected animals, an increase in respiratory rates, body temperature, cough and reduced appetite was observed with mild to moderate severity. infectious virus isolated only from the lower respiratory tract. viral rna was detected in the conjunctiva, nasal mucosa, tonsils, pharynx, trachea, bronchus and lungs. mild to marked interstitial pneumonia with dark red lesions appeared in lungs. seroconversion of neutralizing antibodies began at 7 dpi and increased in titer with time. the development of a transient pneumonia, rapid replication, and tropism of mers-cov for the lower respiratory tract resembled the severity of the disease observed in humans [38, 40, 41] . similarly, the common marmoset was shown to possess the dpp4 receptor [42] . radiographic imaging showed mild to severe bilateral interstitial infiltration and extensive bronchointertitial pneumonia in infected animals. infectious virus was detected in lower and upper respiratory tract tissue and viral rna was detected in nasal mucosa, oropharyngeal swabs, blood, conjunctiva, lymph nodes, gastrointestinal tract, kidney, heart, adrenal gland, liver, spleen, brain and lungs [42] . inoculation of syrian hamsters and ferrets with mers-cov did not result in infection [12, 43] . rabbits may be used as a model to study pathogenesis, transmission, and disease control strategies of mers-cov in vivo as they seroconvert and shed virus after inoculation [44] . in september 2012, a novel coronavirus infection was noted in promed mail [45] . the virus was isolated from the sputum of a 60-year-old saudi male, who was admitted to a hospital with pneumonia and acute kidney injury in june 2012. a few days later, another report appeared describing an almost identical virus detected in a patient in qatar with acute respiratory syndrome and acute kidney injury. the patient had a recent travel history to saudi arabia and then traveled to uk for further medical care [5, 46, 47] . two cases from jordan (april 2012) were retrospectively diagnosed as mers patients. since that time, more than 1542 cases of mers-cov infection have been reported including 544 deaths [48] . the actual number of cases could be higher than those reported [49] . an outbreak of more than 180 confirmed cases including 36 deaths occurred in south korea in may and june 2015. the median age of korean cases were 55 years (range: 16 to 87 years), 60% were men, and 14% were health care professionals. the index case was a 68-year-old male who had recently traveled to several countries in the arabian peninsula [50] . disease control and prevention (cdc), and the ministry of health of saudi arabia (mohsa) as asymptomatic, mild, severely symptomatic, or mortal. cases may be classified into suspected, probable, and confirmed [52, 53] . any person with laboratory confirmation of infection with mers-cov irrespective of clinical signs and symptoms is considered as a confirmed case. who criteria for laboratory confirmation require detection of viral rna or acute and convalescent serology. the presence of nucleic acid can be confirmed by positive results from at least two sequence-specific rrt-pcrs or a single sequence-specific rrt-pcr test and direct sequencing from a separate genomic target [54] . a case confirmation by serological methods requires demonstration of seroconversion in two samples collected at least 14 days apart using at least one screening assay (enzyme-linked immunoassay, immunofluorescence assay) and a neutralization assay. a probable case is defined by the following criteria, a febrile acute respiratory illness as pneumonia or acute respiratory distress syndrome, direct contact with a confirmed mers-cov case and unavailability of mers-cov testing or results being inconclusive for a single inadequate specimen. any person who developed a fever and pneumonia or acute respiratory distress syndrome with a history of travel to countries in or near the arabian peninsula within 14 days before symptom onset or was in contact with a traveler from this region who developed a febrile respiratory illness is considered as a mers-cov suspected case. the who, cdc, and mohsa recommended laboratory diagnostics for mers-cov infection [6, 9, 51, 55, 56] . mers-cov cases must be confirmed by at least two positive qrt-pcr tests on two different specific genomic regions or single positive qrt-pcr with a sequence of another positive genome fragment [57] . the who algorithm for testing mers-cov relies on qrt-pcr and sequencing [58] . available real-time tests include an assay targeting the rna upstream of the e gene (upe) as a highly sensitive screening assay and three confirmatory assays targeting open reading frames (orf 1a and 1b) and/ or n gene. the orf 1a assay is of equal sensitivity to the upe assay. the orf 1b assay is less sensitive but is useful for confirmation. these assays are specific for mers-cov and have not shown cross-reactivity with other respiratory human coronaviruses. for sequencing, two target genes, the rna-dependent rna polymerase (rdrp, present in orf 1b) and n genes are enough to confirm the existence of mers-cov rna in the samples of a patient [57] . several serologic assays including immunofluorescence assays, protein microarray assay, enzyme-linked immunosorbent assay (elisa) have been developed for the detection of mers-cov antibodies [57, [59] [60] [61] . any positive test by one of these assays should be confirmed with a neutralization assay. single serological result may be valuable for definition of probable case and should be followed by further testing for confirmation of mers-cov infection [62] [63] [64] . incubation period of mers-cov infections was studied by assiri et al. in 2013. the median incubation period was 5.2 days (95% ci 1.9-14.7 days) [65] . in another report from france of a secondary case, a patient who shared a room with an infected patient, the incubation period was estimated at 9 to 12 days [66] . in the recent outbreak in south korea during may/june 2015, the median incubation period was 6.3 days [67] . who and cdc recommended that individuals that returned from the arabian peninsula and other affected countries must be evaluated for mers-cov infections up to at least 14 days [68] . clinical features of mers-cov infections range from asymptomatic cases to mildly ill, severe pneumonia, acute respiratory distress syndrome, septic shock and mortal with multi-organ failure (table 1) [64, 65] . many other clinical features such as gastrointestinal symptoms (anorexia, nausea, vomiting, abdominal pain, diarrhea), pericarditis, and disseminated intravascular coagulation were reported [65, 69, 70] . specific clinical conditions (comorbidities) were apparently proportionate with high severity of mers-cov infections. a study by assiri et al. in saudi arabia showed that of a total of 47 patients with mers-cov infection in 2013, 45 (96%) had underlying clinical conditions, including diabetes mellitus (68%), hypertension (34%), chronic cardiac disease (28%), and chronic kidney disease (49%) [65] . this high rate of comorbidities must be interpreted with some caution, since diabetes mellitus is common in saudi arabia, and because approximately half of those 47 were part of an outbreak in a hemodialysis unit, where rates of comorbidities might be high due to chronic kidney disease [65, 71] . in another study, being on dialysis, diabetes mellitus, and age > 50 years was associated with mortality [72] . in this study, testing positive for mers-cov in a plasma sample was a predictor of severe outcome [72] . younger adults and children appeared to be less susceptible to mers-cov infection. only one study described mers-cov infection in children [73] . all of those children were discovered during contact investigations of older patients. only 2 of 11 children developed symptoms of mers-cov infection. these two children had underlying conditions (cystic fibrosis and down syndrome). the other 9 children were asymptomatic. there are few reports of mers-cov infections in pregnant women. a five-month pregnant female developed vaginal bleeding and abdominal pain after one week, then delivered a stillborn infant [74] . another case in the united arab emirates was near term phase, she gave birth to an apparently healthy baby, and died after delivery [52] . mild and asymptomatic mers-cov infections have been reported, a majority of whom were identified among the contacts of patients [62, 75, 76] . in a report from mohsa, more than 3000 contacts of patients were screened using qrt-pcr and seven healthcare workers with mers-cov infection were identified, two of whom were asymptomatic and five of whom had mild upper respiratory tract symptoms [75] . epidemiological and virological studies were conducted in attempts to determine person to person transmission of mers-cov. they studied case clustering in household and hospital outbreaks in the uk, tunisia, italy, and in healthcare facilities in saudi arabia, france, iran, and lately in south korea. those studies provided strong evidence that human-to-human transmission occurs [70, [77] [78] [79] [80] . the number of contacts infected by individuals with confirmed infections, however, appears to be limited [62] , except the outbreak of south korea in may/ june 2015, where most cases were secondary and some cases were tertiary infections [67, 81] . secondary cases often were milder or symptomless [62] . possible modes of transmission include droplet and close contact transmission, air borne transmission, and fomite transmission [82] . the majority of all laboratory-confirmed secondary cases have been associated with healthcare settings [82] . the majority of cases of jeddah, saudi arabia hospital outbreak during the spring of 2014 were acquired through human-to-human transmission due to systematic weaknesses in infection control [76] . secondary transmission rates were assessed within households and the transmission rate was around 4%, suggesting that the actual number of infection is greater than reported [62] . during the outbreak in south korea during may/june 2015, 25 secondary infections were associated with the index case, who was hospitalized from may 15 to may 17 and 11 were tertiary [83] . the median incubation period was six days for secondary cases and six days for tertiary cases. this outbreak also clearly demonstrated roles of "superspreaders," who may be responsible for a high proportion of cases [83] . for instance, a single patient infected more than 70 other people while being treated in the emergency room of a hospital in south korea for three days, 27-29 may 2015. transmissibility and epidemic potential studies of mers-cov revealed that the reproduction number (r 0 ) of patients infected with mers-cov ranged between 0.6 to 0.69 [84, 85] . the finding of an r 0 < 1 suggests that mers-cov does not yet have pandemic potential. other study suggested that r 0 values might reach to 0.8 to 1.3 in the absence of infection control [49] . shedding periods of mers-cov in humans was reported to be long as viruses were detected in lower respiratory samples of symptomatic patients for more than two weeks [86] . at instances, prolonged shedding for 6 weeks was detected in an asymptomatic healthcare worker. these findings raise concerns that asymptomatic persons could transmit infection to others in a silent manner [87] . the majority of cases have occurred in saudi arabia and united arab emirates [88] [89] [90] . many cases have also been reported outside the arabian peninsula in north africa, europe, asia, and north america as shown in table 2 . almost all cases reported outside the arabian peninsula had a travel history to it. the first cluster was in october/november 2012 in four men of the same family in riyadh, saudi arabia, two of whom died [75] . the second cluster was reported in jordan in april 2012 involving 10 healthcare workers exposed to fatal patients. in addition, seven surviving hospital contacts seroconverted suggesting that they had mers-cov infection [91] . the third cluster was reported in uk during january/ february 2013. an english resident had a travel history to saudi arabia and pakistan in january, developed a severe respiratory illness, and tested positive for both mers-cov and h1n1 influenza a, and died in march 2013 after infecting several contacts [92] . a cluster of 43 cases of mers-cov was reported in al-hasa in saudi arabia during april 2013. all those cases were directly linked to human to human contact in the same hospital. there were only two confirmed cases of healthcare workers, and three family members were detected by a survey of over 200 household contacts that visited this hospital [77] . in france, may 2013, an infection of mers-cov was reported in a patient who recently traveled to the united arab emirates. a second case who shared the hospital room with the first case tested positive. the first patient died and the second patient was critically ill. a survey of 100 healthcare workers found no other infections with mers-cov, despite the lack of use of personal protective equipment [70] . a surge in mers-cov cases was reported in saudi arabia and the united arab emirates during march and april 2014 [55, 76] . the majority of cases were associated with hospital-based outbreaks jeddah, riyadh, tabuk, and madinah in saudi arabia as well as in al ain, and abu dhabi in united arab emirates. cases included several healthcare workers, visitors, patients, and ambulance staff. person to person transmission was confirmed in > 75% of cases. the majority of infected health care workers developed mild symptomatic or asymptomatic infection, but about 15% had severe illness or died [93] . the recent outbreak of south korea occurred in may 2015. the index case was a man who had recently traveled to bahrain, the united arab emirates, saudi arabia, and qatar [55] . as of late july 2015, > 180 secondary cases were reported including 36 death and many cases had been reported among household and hospital contacts [55, 67] . in china, one case occurred in a man who traveled to china from korea following exposure to two relatives with mers-cov infection [55] . in spite of reporting of mers-cov infections throughout the year, some evidence on disease seasonality occurred. the first identified cases of mers-cov infection were reported in april and june 2012 [5, 46, 47] . a high increase in cases was reported in april and may 2013 followed by a surge in case reporting in april and may 2014. increase in case reporting in march to may 2013 were attributed to infection from infected young camels [94, 95] sources and modes of transmission of mers-cov are still unclear. initially, a bat origin of mers-cov was suggested based on the relation of genome sequences between mers-cov and bat coronaviruses [96] . cell tropism studies showed that both bat coronavirus hku4 and mers-cov shared the same cell type receptors, dpp4 [4, 7, 75] . mers-cov grows readily in several bat-derived cell lines [14] . there is no evidence for direct or indirect transmission of mers-cov from bats to humans. virological studies performed in europe, africa, and asia, including the middle east, have shown that coronavirus rna sequences are found frequently in bat feces. some of the sequences were closely related to mers-cov sequences [97] [98] [99] . in a survey from saudi arabia, 823 fecal and rectal samples were tested by pcr for mers-cov, many coronaviruses sequences were detected [97] . most of the detected sequences were unrelated to mers-cov, but one sequence of 190 nucleotide in the rna-dependent rna polymerase (rdrp) gene had a 100% identity with a mers-cov. this sequence was detected from feces of a taphozous perforatus bat captured near the home of the index saudi patient. uncommon contact of humans with bats indicates that bats are not the intermediate host of mers-cov transmission but may be the reservoir of the virus [100] . dromedary camels (camelus dromedarus) appear to be the source of mers-cov. other animals like sheep, goats, and cows tested negative to anti-mers-cov antibodies. camel sera from oman, canary islands, and egypt were positive for anti-mers-cov antibodies in about 100%, 14%, and > 90% of the samples respectively [63, 101, 102] . retrospective studies on archived human sera showed no evidence of infection with mers-cov before 2012 [103] , but anti-mers-cov antibodies were detected in archived camel sera in saudi arabia in 1993 [95] , and united arab emirates in 2003 [104] , indicating circulation of mers-cov in camels for many years. bactrian camels in mongolia tested negative for mers-cov antibodies [105] . serologic studies from around the middle east suggested that camels are one of the sources of mers-cov as > 90% of adult camels tested positive and had high titers of antibodies. seropositivity was different in juvenile camels and was usually lower than in adults. these results suggested that mers-cov infections in camels occurred in young ages followed by frequent boosting [94, 95, 102, 104] . camels in other parts of the world, far from the middle east like in europe, australia, and the americas do not have mers-cov antibodies and have no evidence of infection [106] . table 3 summarizes camel serologic studies. in a study aimed to evaluate virus infectivity and shedding in camels, three adult dromedary camels were inoculated with mers-cov intratracheally, intranasally, and conjunctivally. those camels shed large quantities of virus from the upper respiratory tract and infectious virus was detected in nasal secretions for 7 days post-inoculation and viral rna for up to 35 days post-inoculation [113] . human infections with mers-cov were linked to camels. the first evidence was a study in saudi arabia in which the mers-cov full genome sequences of isolates from a man with fatal infection and from one of his camels were identical. this patient had a direct contact with his deceased camels some days before the onset of symptoms. these results suggested that mers-cov can infect dromedary camels and can be transmitted from them to humans by direct close contact [86] . in other studies, phylogenetic analyses of camel and human isolates of the mers-cov genome demonstrated that the viruses were highly identical or in some cases were similar to each other [107, 108, 114] . seroepidemiological studies shown low prevalence of mers-cov antibodies in humans in saudi arabia [103, 115] . a survey of 10 009 individuals representative of the general population of saudi arabia resulted in 15 seropositive subjects (0.15%), however, seropositivity increased 15-23 folds in camel-exposed individuals [78] . in a separate report, 7 of 87 camel shepherds and 140 slaughterhouse workers (3.1%) tested positive for mers-cov antibodies [103] . an overview of mers-cov transmission routes is illustrated in fig.2 . the development of an effective vaccine is critical for prevention of a mers-cov pandemic. some investigators have indicated that the rbd protein of mers-cov s protein is a good candidate antigen as a subunit vaccine. various rbd fragments showed the highest dpp4 binding affinity and induced the highest-titer of igg ab and neutralizing ab in mice and rabbits [17, 18, [116] [117] [118] [119] [120] . a robust neutralizing antibody response was elicited in balb/c mice against mers-cov after immunization with purified full s protein nanoparticles produced in sf9 cells infected with specific recombinant baculovirus containing the s gene [121] or a recombinant human adenoviral vectors (rad5 or rad41) containing the s or s1 genes [122, 123] . vaccinia ankara was encoded with full s protein and inoculated to balb/c mice that developed high levels of neutralizing antibodies and had induction of humoral and cell-mediated immunity [124, 125] . another study using ad5-hdpp4-transduced balb/c mice immunized with venezuelan equine encephalitis virus replicon particles containing s protein elucidated a reduction of viral titers to nearly undetectable levels and increased neutralizing antibodies [35] . recently, wang et al. developed two candidate vaccines, a subunit (full s and s1 protein fraction) and a dna vaccine (full s and s1 gene in a mammalian vrc8400 vector). the vaccine containing the full s dna and s1 protein was the most efficacious in mice and rhesus macaques [126] . using antibodies to deter mers-cov infection appears to have some promise. transfer of sera containing anti-mers-cov-s protein to or seropositive camel sera to ad5-hdpp4-transduced mice accelerated virus clearance, inhibited virus attachment, and reduced weight loss [35, 37, 127] . recently, corti et al. successfully isolated monoclonal antibodies from serum obtained from a mers-cov survivor after 200 days of infection [128] . transduced ad5-hdpp4 balb/c mice were immunized with 15 mg/kg of the mab and showed decreased lung [105] viral titers, no weight loss, and decreased peribronchial lymphoid infiltration [128] . no approved antivirals for use against mers-cov infection are yet available. the first approach performed when a new unknown virus like mers-cov emerges is testing drugs used as antiviral for similar viruses [29, 129, 130] . type i interferons and ribavirine combination exhibited acceptable results in cell culture and rhesus macaques by decreasing the host inflammatory response, replication of virus, and improved clinical outcome [129, 131, 132] . a human cohort study in saudi arabia showed that treatment with combination of ribavirin and interferon-α2b to 5 did not improve clinical outcomes but this may have been due to late treatment or due to the immunocompromised state of the patients [133] . in a retrospective study of 20 mers-cov infected patients treated with ribavirin and interferon α-2a, results showed 14-day and 28-day survival was improved by 70% and 28% in the treated group as compared to an untreated group [134] . the second approach is screening of approved drugs with known safety profiles and transcriptional signatures in different cell lines. several drugs, including antiparasitics, neurotransmitters, antibacterials, inhibitors of clathrinmediated endocytosis estrogen receptor, lipid or sterol metabolism, protein processing, and dna synthesis or repair were tested on culture cells [119, [135] [136] [137] [138] [139] . lopinavir-ritonavir combined with pegylated interferon and ribavirin therapy showed improved outcomes in infected marmosets [140] . the third approach involves in vitro inhibition of s protein to block virus entry into host cells using designed antiviral peptides targeting the hr2 domain of the s2 subunit of the mers-cov and preventing the interaction between the hr1 and hr2 domains required for the formation of the heterologous six-helix bundle in viral fusion core formation [22, 23] . other drugs that act as inhibitors for viral proteases and helicase to suppress mers-cov infection were tested [141] [142] [143] [144] [145] . other investigators studied inhibition of mers-cov infection by competitive inhibition of dpp4 cell receptor using compounds such as sitagliptin, vildagliptin, and saxagliptin [19, 146] . more than three years have passed since the first detection of mers-cov human infection and the virus, uncontrolled, continues to cause major outbreaks in the middle east. the recent outbreak in korea demonstrated that a single index case can lead to 185 more infections in a short period of time, hence raising questions about the accuracy of the number of cases being reported in the middle east. furthermore, the korean outbreak confirmed the high fatality rate of mers-cov infection as being true rather than overestimated in case only the more severe cases are detected. in all, public health, veterinary health, and research efforts need to be consolidated in order to answer the following high priority questions: -what is the true extent of human infection with mers-cov? -what antivirals and vaccines are to be used in humans? -what infection control measures are needed in healthcare settings to prevent nosocomial outbreaks? -what measures need to be in place in order to prevent zoonotic infections from camels? -is it possible to control the virus in the camel population and if so, how? -are there other animal species involved in the mers-cov transmission cycle? a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease coronavirus as a possible cause of severe acute respiratory syndrome discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east 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important differences with sars coronavirus antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis testing of middle east respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus the newly emerged sars-like coronavirus hcov-emc also has an "achilles' heel": current effective inhibitor targeting a 3c-like protease inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody mahmoud m. shehata, mokhtar r. gomaa, mohamed a. ali, and ghazi kayali declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. key: cord-341795-zbqfs77n authors: sikkema, r. s.; farag, e. a. b. a.; islam, mazharul; atta, muzzamil; reusken, c. b. e. m.; al-hajri, mohd m.; koopmans, m. p. g. title: global status of middle east respiratory syndrome coronavirus in dromedary camels: a systematic review date: 2019-02-21 journal: epidemiol infect doi: 10.1017/s095026881800345x sha: doc_id: 341795 cord_uid: zbqfs77n dromedary camels have been shown to be the main reservoir for human middle east respiratory syndrome (mers) infections. this systematic review aims to compile and analyse all published data on mers-coronavirus (cov) in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of infections in dromedary camels. we included original research articles containing laboratory evidence of mers-cov infections in dromedary camels in the field from 2013 to april 2018. in general, camels only show minor clinical signs of disease after being infected with mers-cov. serological evidence of mers-cov in camels has been found in 20 countries, with molecular evidence for virus circulation in 13 countries. the seroprevalence of mers-cov antibodies increases with age in camels, while the prevalence of viral shedding as determined by mers-cov rna detection in nasal swabs decreases. in several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms as well as imported camels vs. locally bred camels. some studies show a relatively higher seroprevalence and viral detection during the cooler winter months. knowledge of the animal reservoir of mers-cov is essential to develop intervention and control measures to prevent human infections. middle east respiratory syndrome (mers) is a highly fatal respiratory tract disease in humans that was first detected in 2012 in the kingdom of saudi arabia (ksa) [1] . after its first detection, mers-coronavirus (mers-cov) was being reported in human patients across the arabian peninsula, with occasional travel-related cases in other continents. as of the end of march 2018, a total of 2189 human laboratory-confirmed cases from 27 countries have been reported to the world health organisation (who), including 782 associated deaths [2] . dromedary camels (camelus dromedaries) have been shown to be the natural reservoir from where spill-over to humans can occur [3, 4] . human-to-human infection is also reported frequently, especially in healthcare settings [5] . sustained human-to-human transmission outside of hospital settings has not been shown yet [6] . direct or indirect human contact with camels has resulted in repeated introductions of mers-cov into the human population [7] . it has been suggested that camels may have acquired mers-cov from a spill-over event from a bat reservoir, but evidence for that remains inconclusive [8] . infections with mers-cov generally are thought to be mild or inapparent in camels [9] , and are therefore of low economical or animal welfare significance. this systematic review was done to compile and analyse all published data on mers-cov in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of mers-cov infections in dromedary camels as a basis for the design of intervention and control measures to prevent human infections. on 2 may 2018, a literature search on pubmed was performed, using the terms 'middle east respiratory syndrome coronavirus' and 'mers-cov'. using the term 'mers' did not result in any additional articles that fit the scope of this review. only articles published in english were included. two reviewers individually selected all original research articles containing laboratory evidence of mers-cov infections in dromedary camels in the field. articles that were mentioned in food and agriculture organization (fao) updates [10] or in the references of included publications, but did not appear in the pubmed search were added. subsequently, abstracts, follow-up studies of mers-cov-positive camels and genome studies without prevalence data were excluded from the analysis. data on variables such as year of sampling, country, region, age, sex and animal origin were extracted and analysed. for each variable, the number of positive camels, total number of camels tested and the median percentage positivity was calculated. data from experimental infection studies were not included in this analysis, but they were included in the review to provide additional information and context to the field studies. additional information on the distribution and trade of dromedary camels was collected from references in the publications on mers-cov in camels and extracted from official fao and world organisation for animal health (oie) databases [11, 12] . the additional literature on camel trade was collected in a less systematic way from pubmed. the literature search resulted in a total of 53 papers (fig. 1 ). forty-three research papers described the results of crosssectional studies in dromedary camel populations, six papers described outbreak investigations, including an analysis of camel samples, and four papers described longitudinal studies. in total, 33 papers describe camel studies in the middle east, 13 studies investigated camels from africa and the remaining seven surveys were from spain, australia, japan, bangladesh and pakistan (table 1) . most recent fao statistics estimate the world population of camel to be around 29 million [11] , of which approximately 95% are dromedary camels [13] . however, it is believed that the true population size is even larger due to inaccurate statistics and feral camels, such as the feral dromedary camel population in australia that is estimated to be around 1 million [14] . over 80% of the camel population lives in africa. the main camel countries are chad (6 400 000), ethiopia (1 200 000), kenya (2 986 057), mali (1 028 700), mauritania (1 379 417), niger (1 698 110), sudan (4 830 000), somalia (7 100 000) and pakistan (1 000 000) [12] (table 2) . a large number of camels are being transported from the horn of africa to the middle east each year. these are mainly meat camels coming from the east of africa going to egypt, libya and the gulf states, and sudanese camels that are being imported into the middle east to participate in camel racing competitions [15] . for example, the fao reported that somalia exported 77 000 camels in 2014 [16] . the largest camel market in africa is the birqash market near cairo (egypt), where camels from sudan and ethiopia are most common, but trade routes include animals from chad, somalia, eritrea and kenya [17] . imported camels are usually quarantined for 2-3 days at the border before they are allowed to enter egypt [17] . most somali and sudanese camels that are exported to the ksa are shipped from the ports of berbera and bosaso in north somalia to the ksa ports of jizan and jeddah [15] . in general, only minor clinical signs of disease have been observed in animals infected with mers-cov and most mers-cov infections do not appear to cause any symptoms [9] . disease symptoms that have been described after experimental and field infections are coughing and sneezing, respiratory discharge, fever and loss of appetite [18] [19] [20] . although mers-cov rna can be detected in several organs after experimental infection, in studies of natural infectious virus it has only been detected in the tissues of the upper and lower respiratory tract and regional lymph nodes of the respiratory system in part of the infected camels. histologically, a mild-to-moderate inflammation and necrosis could also be seen on the upper and lower respiratory tract. no viral antigen or lesions were detected in the alveoli. histopathological examination showed that the nasal respiratory epithelium is the principal site of mers-cov replication in camels [18, 21] . in one study investigating experimental infection of camels, mers-cov shedding started 1-2 days post-infection (dpi). in that study, infectious virus could be detected until 7 dpi, and viral rna until 35 dpi in nasal swab samples and, in lower amounts, in oral swab samples [18] . no infectious virus or viral rna was detected in faecal or urine samples [18] . viral rna detection in nasal, but also rectal swabs of camels after experimental infection until day 14, has been confirmed in a recent vaccine study [21] . in the field surveys included in this review, mers-cov rna has been described in rectal swab samples, although other field studies report negative results [3, [22] [23] [24] and when viral rna can be detected, the positivity rate of rectal swabs is lower compared with nasal swab samples [19, [25] [26] [27] . oral swabs are usually negative or show a lower positivity rate even when nasal swabs test positive for mers-cov rna [3, 19, 26] . some studies have reported mers-cov rna in milk samples [27, 28] . longitudinal studies of camel herds show that pcr results of nasal swabs can remain positive after 2 weeks [27, 29] . when an interval of sampling of 1 or 2 months was maintained, nasal swabs become negative for viral rna in the next sampling round [24, 30] . mers-cov infections have also been detected in camels with mers-cov antibodies, both in calves with maternal antibodies as well as older camels that had already acquired antibodies from a previous infection. however, virus replication and thus the virus load is generally lower in infected seropositive animals compared with seronegative camels [19, 21, 23, 24, 30, 31] . little is known about the longevity of antibody titres after infection from longitudinal studies. a study following camels on a closed farm found that neutralizing antibodies remained consistent during a year [30] , while other studies found that antibody titres rapidly drop by 1-4-fold within a period often as short as 2 weeks [24, 27] . the first evidence of mers-cov in camels described so far is the detection of antibodies to mers-cov in camel sera from somalia and sudan from 1983 of which 81% tested positive [32] . additional serological evidence of the widespread presence of mers-cov infection in camels, included in this review, has been found in 18 additional countries: bangladesh, burkina faso, egypt, ethiopia, iraq, israel, jordan, kenya, ksa, mali, morocco, nigeria, oman, pakistan, qatar, spain, tunisia and the uae (fig. 2 ). in addition, promed mail reported that virus-positive camels had been found in kuwait and iran, the latter reportedly in imported animals (archive number 20140612.2534919 and 20141029.2912385). in 11 countries, serological findings were complemented with the finding of viral rna in dromedary camels: burkina faso, egypt, ethiopia, iraq, jordan, ksa, morocco, nigeria, oman, qatar and the uae. investigations of mers-cov circulation amongst dromedary camels in australia, japan, kazakhstan, usa and canada did not find any proof of mers-cov circulation. all countries where mers-cov circulates in the camel population, with the exception of spain (canary islands), pakistan and bangladesh, are located in the middle east or africa [4, 33] . one out of 17 camels that had mers-cov antibodies in bangladesh was born in bangladesh, 16 others were imported from india [34] . however, there have not been any additional reports of mers-cov in camels in india. there is no record of foreign origin of the seropositive camels from pakistan [35] . moreover, in previous studies there had already been evidence of seropositive camels that originate from pakistan [37, 58] . when combining serology data from all papers included in this review, the overall median seroprevalence of camels in africa is 81% (6106/8526; range 28-98%), compared with a median seroprevalence of 93% (3230/3846; range 53-100%) in camels from the middle east. based on viral shedding studies from african countries, the median rate of viral shedding was 5% (1108/6318; range 1-15%), compared with 12% in camels from the middle east (1191/14902; range 0-100%). the seroprevalence of mers-cov antibodies increases with age in camels, while the fraction of camels that test positive for mers-cov rna in their nasal swabs decreases with age [17, 31, 36, 38, 39] . when all serological results of papers that included sufficient age information is combined, the median seroprevalence of camels aged under 2 years is 52% (992/1972; range 0-100%), while the age groups 2-5 years (702/924; range 30-100%) and over 5 years old (1226/1370; range 0-100%) had a combined median seroprevalence of 97%. in the virological studies reporting age breakdown, the median rate of nasal shedding in 0-2 years old camels was 34% (718/2612; range 0-100%) of cases, compared with 2% (91/1142; range 0-100%) in camels older than 2 years. some individual studies show a significantly higher seroprevalence in female camels compared with males [27, 39] , while others show the opposite [38] or do not find any significant difference [17, 35] . similar disagreeing results are published for the presence of mers-cov rna in male vs. female camels [17, 27, 38, 39] . in the studies in this review where sex of camels was recorded, a total of 4810 serum samples from female camels and 3458 samples from male camels were collected and analysed for mers-cov antibodies, compared with 2007 vs. 2505 nasal swabs for viral rna testing. approximately three times more female camels were sampled at farms, while male camels were in the majority in studies that looked at mers-cov prevalence of camels at slaughterhouses, live animal markets and quarantine areas. the overall median seroprevalence of male and female camels in our review is 50% and 67%, respectively (range 0-100%; excluding results from israel and kazakhstan). the median percentage of presence of viral rna is 18% in nasal swabs of male camels (range 0-21%) compared with 9% in female camels (range 0-100%), in our review. in several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms [17, 22, 27, 34] . combining serological laboratory results of camels in our review with sufficient background information with regard to the sampling location does not result in the same pattern, with a median seroprevalence of 84% (5632/8115; range 0-100%; excluding australia and spain) in camels from farms and 80% (943/1005; range 28-98%) in the camel population sampled at markets and quarantine facilities. studies in egypt found a significantly higher pcr positivity rate in camels sampled in abattoirs or quarantine facilities, but these results could not be confirmed by other papers in this review [17, 27] . when comparing differences in seroprevalence or virus rnapositive rate in nomadic vs. sedentary camel herds, some authors did not find a statistical difference between the two herd management types [39, 40] , while others found some evidence of higher seroprevalences in nomadic herds [27, 36] . one study in kenya looked at the differences between herds with different levels of isolation, and did not find significant differences in mers-cov antibody levels [40] . most studies that compared local camels with imported camels suggested that imported camels are seropositive for mers-cov more often [9, 17, 27, 34, 41] , although not all differences were significant. two studies in egypt found a significantly higher rna positivity rate in imported camels from east africa compared with domestically bred camels [17, 27] , while another study executed in the ksa found a significantly higher number of mers-cov rna-positive results amongst local camels vs. camels from sudan and somalia [22] . although mers-cov was detected almost year-round in camels, some studies show a relatively higher seroprevalence and viral detection during the cooler winter months [17, 20, 27, 38] . mers-cov antibodies have been detected in llamas and alpacas in israel and in alpacas in qatar [42, 43] . to date, no mers-cov antibodies or viral rna have been detected in bactrian camels [4, 37, [44] [45] [46] [47] (table 1 and table 3 ). swine, goats and horses that were included in the field surveys in our review all tested negative for mers-cov rna and antibodies [4, 17, 31, [48] [49] [50] [51] [52] . mers-cov antibodies were detected in two studies in sheep in egypt and qatar, although in very low numbers [17, 51] . however, most surveys that investigated sheep did not find evidence of mers-cov infection or exposure [4, 23, 29, 31, 34, [48] [49] [50] [51] 53] . the publications in this review show that the mers-cov mainly circulates in dromedary camel populations in the middle east and part of africa, and has been infecting dromedary camels in africa for more than three decades. antibodies have also been found in arabic camel sera from the early 90s [31, 32] . however, mers-cov was discovered until 2012, after the first human cases appeared [1] , which is probably due to the minor clinical symptoms of mers-cov infections in camels [18] . most camel surveys were conducted in the middle east and some northern and eastern african countries, but significant data gaps currently still exist in the north and west of africa, in countries that have camel populations of 100 000 to more than a million animals, such as algeria, libya, mauritania and niger. even less is known about the central asian region. some evidence of mers-cov circulation in camels of pakistan and bangladesh was recently published, but data is lacking from afghanistan and india. knowledge on the presence of mers-cov in the animal reservoir is a crucial first step to assess whether mers-cov could be a relevant public health threat in these regions. mers-cov infections are mainly detected in calves and young camels [30, 31] . the research included in this review shows that the igg positivity rate increases gradually in dromedary camels of increasing age while the mers-cov rna detection rate decreases. maternal igg antibodies in camels are acquired through the intake of colostrum during the first 24 h post-parturition. after 24 h, antibody levels in the dam's milk decrease rapidly [54] . one study showed that maternal antibodies in calves peak at 7 days postparturition and decline in the following 6 months. after 5-6 months, over half of the calves did not have maternal neutralizing antibodies in their serum any longer [30] . however, in other field studies, the titre of mers-cov-specific antibodies is still low at 1 month of age and increases with age in dromedary calves [27, 55] . a lower or undetectable antibody levels in young camels is likely to explain the higher mers-cov rna detection rate. in adult camels, a much higher mers-cov seroprevalence can be found, which is probably due to a long-lasting immune response against a mers-cov infection or multiple re-infections with mers-cov. immunity is not sterilizing, as mers-cov infection and shedding have also been shown in adult camels that have mers-cov antibodies [19, 21, 23, 24, 30, 31] . several articles have analysed seroprevalence and virus shedding data in relation to factors, other than age, that may explain differences in seroprevalence and mers-cov rna-positive rate in camels, such as sex, sampling location, herd characteristics and animal origin. our review shows that there is considerable heterogeneity in results. in addition, comparison between studies is difficult given the lack of standardisation of study designs. a key factor to consider when comparing studies is the difference in distribution of male and female camels amongst different disciplines of camel husbandry. females are mainly used for milking and reproduction. as a result, they often stay at farms. male camels, especially of young age (<1 year old), are the predominant sex in slaughterhouses and amongst camels used for transport [39, 56] . this also influences the risk profile of acquiring a mers-cov infection. female camels are in closer contact with calves, who are more susceptible to infection and shed virus in higher quantities compared with older camels [30] . on the other hand, meat and transport camels (predominantly male) travel more, leading to increased contact with other camels and camel herds, and therefore a higher chance of exposure to mers-cov. some papers in this review suggest that there is a generally lower infection rate of domestically bred camels and camels on farms compared with imported camels and camels on animal markets or in quarantine facilities. this may be explained by the same increased contact rate and mixing of camel herds, leading to an increased chance of mers-cov exposure and spread. the increase in mers-cov circulation in winter and spring can have multiple explanations. firstly, the winter is the calving season [10] , which leads to a larger proportion of young animals that usually have a higher number of mers-cov infections and virus excretion. moreover, in winter season, there is a major increase of camel and human movements due to camel racing competitions, camel breeding, trading and movements to grazing grounds, which increases the chance of virus spread. additionally, cooler temperatures may facilitate coronavirus survival in the environment [57] . in experimental studies, llama's and alpaca's are shown to be susceptible to infection with mers-cov [58, 59] , which was confirmed by two papers in our review, describing serologically positive llamas and alpacas in israel and alpacas with mers-cov neutralizing antibodies in qatar [42, 43] . in experimental settings, animal-to-animal transmission has been shown for alpacas, making them a possible risk population for human infections [58] . two studies in our review also found anti-mers-cov antibodies in sheep [17, 51] but experimental inoculation of sheep did not result in mers-cov replication or antibody development [59, 60] . however, the dpp4 receptor, the entry receptor for mers-cov, is present in sheep tissues, making it possible for the virus to bind to the sheep respiratory tract which may explain the finding of mers-cov antibodies [61] . pigs also express the dpp4 receptor in their respiratory tract, and viral replication in experimental settings has been shown for pigs, but no antibodies or mers-cov rna have been found in pigs during field surveys [48, 59] . this may be explained by the limited viral shedding in pigs and the absence of animal-to-animal transmission [62, 63] . we show that dromedary camels are present in large parts of the african and asian continent, and that mers infections in dromedary camels are widespread. however, human infections due to spill-over from the dromedary camel reservoir have not been reported in africa [10] . several explanations for the difference in human cases between the arabian peninsula and africa have been suggested, such as differences in cultural habits, camel husbandry, prevalence of comorbidities, under detection or genetic factors in the local population [64] . moreover, west african viruses were found to be phylogenetically and phenotypically distinct from the mers-cov viruses that caused human disease in the middle east [65] . increased knowledge on the animal reservoir of mers-cov needs to be combined with research on mers prevalence and risk factors in humans to assess the true public health risk. moreover, the absence of human disease, combined with the mild symptoms in camels, caused by mers, will likely have a negative effect on the willingness to implement interventions and the cost-effectiveness of possible interventions in some areas. since the discovery of mers-cov in 2012, the dromedary camel has been identified as the animal reservoir of human infections with the mers-cov. however, the exact route of human primary infections is still unknown. moreover, the scale of the spread and prevalence of mers-cov in the camel reservoir is not fully known yet since there is still a lack of mers-cov prevalence data in some countries that harbour a very significant proportion of the world camel population. however, knowledge of the animal reservoir of mers-cov is essential to develop intervention and control measures to prevent human infections. prospective studies that include representative sampling of camels of different age groups and sex, within the different husbandry practices, are needed to fully understand the patterns of mers-cov circulation. such studies are important as they may give more information on critical control points for interventions to reduce the circulation of mers-cov and/or exposure of humans. author orcids. r. s. sikkema, 0000-0001-7331-6274 financial support. this study was financially supported by the european commission's h2020 programme under contract number 643476 (http:// www.compare-europe.eu/). none. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers situation update march middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east 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collected according to local customs from dromedary camels epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate time course of mers-cov infection and immunity in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia mers coronavirus neutralizing antibodies in camels presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands middle east respiratory syndrome coronavirus antibodies in dromedary camels serologic evidence for mers-cov infection in dromedary camels antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia risk factors for mers coronavirus infection in dromedary camels in burkina faso, ethiopia, and morocco serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels absence of mers-coronavirus in bactrian camels absence of middle east respiratory syndrome coronavirus in bactrian camels in the west inner mongolia autonomous region of china: surveillance study results from absence of middle east respiratory syndrome coronavirus in camelids middle east respiratory syndrome coronavirus infection not found in camels in japan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan serologic assessment of possibility for mers-cov infection in equids dromedary camels in northern mali have high seropositivity to mers-cov. one health studies on the supply of immunoglobulin g to newborn camel calves (camelus dromedarius) acute middle east respiratory syndrome coronavirus infection in livestock dromedaries the camel today: assets and potentials stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus livestock susceptibility to infection with middle east respiratory syndrome coronavirus inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 middle east respiratory syndrome coronavirus experimental transmission using a pig model domestic pig unlikely reservoir for mers-cov mers-cov antibodies in humans mers coronaviruses from camels in africa exhibit region-dependent genetic diversity genetic diversity and phylogeographic structure of bactrian camels shown by mitochondrial sequence variations evidence for camel-to-human transmission of mers coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate phylogenetic analysis of merscov in human and camels in iraq no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons key: cord-260518-mswb3q67 authors: zumla, alimuddin; dar, osman; kock, richard; muturi, matthew; ntoumi, francine; kaleebu, pontiano; eusebio, macete; mfinanga, sayoki; bates, matthew; mwaba, peter; ansumana, rashid; khan, mishal; alagaili, abdulaziz n.; cotten, matthew; azhar, esam i.; maeurer, markus; ippolito, giuseppe; petersen, eskild title: taking forward a ‘one health’ approach for turning the tide against the middle east respiratory syndrome coronavirus and other zoonotic pathogens with epidemic potential date: 2016-06-15 journal: int j infect dis doi: 10.1016/j.ijid.2016.06.012 sha: doc_id: 260518 cord_uid: mswb3q67 the appearance of novel pathogens of humans with epidemic potential and high mortality rates have threatened global health security for centuries. over the past few decades new zoonotic infectious diseases of humans caused by pathogens arising from animal reservoirs have included west nile virus, yellow fever virus, ebola virus, nipah virus, lassa fever virus, hanta virus, dengue fever virus, rift valley fever virus, crimean-congo haemorrhagic fever virus, severe acute respiratory syndrome coronavirus, highly pathogenic avian influenza viruses, middle east respiratory syndrome coronavirus, and zika virus. the recent ebola virus disease epidemic in west africa and the ongoing zika virus outbreak in south america highlight the urgent need for local, regional and international public health systems to be be more coordinated and better prepared. the one health concept focuses on the relationship and interconnectedness between humans, animals and the environment, and recognizes that the health and wellbeing of humans is intimately connected to the health of animals and their environment (and vice versa). critical to the establishment of a one health platform is the creation of a multidisciplinary team with a range of expertise including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral phylogeneticists, and researchers to co-operate, collaborate to learn more about zoonotic spread between animals, humans and the environment and to monitor, respond to and prevent major outbreaks. we discuss the unique opportunities for middle eastern and african stakeholders to take leadership in building equitable and effective partnerships with all stakeholders involved in human and health systems to take forward a ‘one health’ approach to control such zoonotic pathogens with epidemic potential. benefit the large majority of affected people. some foreign aid workers and researchers were not familiar with local cultural and medical services norms and aroused local anxieties. 10 the evd epidemic highlighted the need for developing more comprehensive local, national, international, and global surveillance, as well as epidemic and outbreak preparedness response infrastructures. multiple animal, human, and environmental factors are obviously playing a critical role in the evolution, transmission, and pathogenesis of zoonotic pathogens, and these require urgent definition to enable appropriate interventions to be developed for optimal surveillance, detection, management, laboratory analysis, prevention, and control in both human and animal populations. an important need exists for establishing long-term, sustainable, trusting and meaningful and equitable collaborations between the animal, human, ecosystem, and environmental health sectors at the local, national, and international levels. these should include sustainable political and funder support for developing human and laboratory capacity and training that enables effective human-animal health cooperation leading to proactive surveillance, early detection of potential pandemic pathogens, and rapid initiation of public health prevention and control guidelines and interventions. whilst a long list of pathogens with epidemic potential are on the radar of the world health organization (who), 2 ideally 'prevention is better than cure' and new pathogens should be dealt with at the animal source, tackling the drivers and triggers of pathogen evolution and emergence. this requires close cooperation between human and animal health systems and an appreciation of human impacts on the environment at all levels and easy access to adequate laboratory facilities. on december 10, 2015 an expert panel convened by who prioritized a list of emerging pathogens ''considered likely to cause severe outbreaks in the near future, and for which no, or insufficient, preventive and curative solutions exist''. 11, 12 the list of the top 10 includes the new viral zoonotic pathogen of humans mers-cov, 13, 14 which was first isolated from a patient who died of a severe respiratory illness in a hospital in jeddah, saudi arabia in june 2012. 15 the emergence of mers-cov in 2012 15 was the second time (after sars-cov 16 ) that a highly pathogenic coronavirus of humans emerged in the 21 st century. 17 a strong link between human cases of mers-cov and dromedary camels has been established through several studies. [18] [19] [20] [21] [22] [23] [24] [25] [26] mers-cov is endemic in the camel populations of east africa and the middle east 21, 25, 26 and presents a constant threat to human health in both regions. retrospective studies using stored serum from different geographical locations have indicated that mers-cov has been circulating for several decades. 25 as of may 1, 2016, there have been 1733 laboratoryconfirmed cases of mers reported to the who, 27 with a mortality of 34% (628 cases died). whilst most mers cases have been reported from the middle east (a large proportion from saudi arabia), mers cases have been reported from 27 countries in all continents. 27 the who has held nine meetings of the emergency committee (ec) for mers-cov. 28 since evidence of sustained human-to-human transmission of mers-cov in the community is lacking, the who currently does not recommend travel restrictions to the middle east. however, mers-cov remains a major global public health threat with continuing reports of new human mers cases in saudi arabia, where millions of pilgrims from over 184 countries travel throughout the year. 29 furthermore, a more intensive farm-based camel livestock system has emerged and there is a large, wellestablished trade in camels between countries at the horn of africa and countries in the middle east. this has increased significantly, particularly following the lifting of the ban on live animal imports from somalia by saudi arabia in 2009/2010. somalia now exports some five million live animals every year to the gulf arab states (including 77 000 camels), making it the single biggest exporter of live animals in the world. the positive experience of reviving somalia's livestock export industry through increased investment in animal disease prevention and control strategies highlights how effective the 'one health' approach can be. most of the african countries do not have the resources, expertise, or capacity, including laboratory facilities, to have active surveillance for mers-cov in place. in light of this, the need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa has been highlighted previously. 30 such an initiative could be supported through investments by countries that import large numbers of camels and other livestock from the region. the epidemic potential of mers-cov was recently illustrated by a large outbreak in hospitals in seoul, the republic korea, in mid-2015: mers-cov was imported by a traveller to the middle east (an agriculture businessman), resulting in 184 mers cases with 33 deaths. 31 the first case was reported on may 20, 2015 and over the ensuing 3 weeks, the number of secondary, tertiary, and perhaps quaternary cases of mers from this single patient rose rapidly, resulting in the largest mers case cluster occurring outside the middle east. the unprecedented outbreak was attributed to poor infection control measures at the hospitals. 30 sequencing studies of the mers-cov isolate showed genetic recombination of mers-cov in the case exported from korea to china. 32 however, recombination is a frequent event in mers-cov and the korean outbreak is unlikely to represent a special form of the virus. nonetheless, the potential evolution of mers-cov into a more virulent form needs to be monitored closely. research on sequencing seems to have stagnated and there have been no further sequences published from new human mers cases reported from the middle east. furthermore, the genetic evolution of mers-cov strains infecting humans over the past year remains unknown. there is an urgent need for more sequencing studies on mers-cov evolution in camels and humans, with the development of appropriate local capacity for these studies. the kingdom of saudi arabia has kept proactive watchful mers-cov surveillance with regular reports to the who of mers-cov cases. 33 the who and ministries of health of middle eastern countries continue watchful surveillance of the mers-cov situation, and the watchful anticipation is that mers-cov may disappear with time like sars-cov. however, with the continuing, regular reports of community cases of mers-cov from saudi arabia, 27 there are no signs of this happening in the near future and lessons must be learnt from the korean outbreak. 34 whilst there is a growing camel livestock industry in the region, elimination of the virus is unlikely in the short term. several animal, human, and environmental factors are obviously playing a critical role in the repeated movement of mers-cov from camels to humans. the disease ecology remains largely unknown. urgent definition is required to enable appropriate interventions to be developed for optimal surveillance, laboratory detection, management, prevention, and control in both human and animal populations. whilst several ad hoc research studies have been conducted and findings published over the past 4 years, more comprehensive investments in tackling mers-cov have not been forthcoming. there remain huge knowledge gaps on mers-cov. much of the information that we have about the source of mers-cov infections is based on small local studies and it is difficult to develop general country-wide policies without a clear understanding of the zoonotic problem. questions remain, for example are new local mers outbreaks in saudi arabia always seeded by the same type of human exposure to camels? are there particular regions of africa that provide infected camels to saudi arabia? or is there a general risk from all regions? is there a way to efficiently control the entry of infected camels? are animal vaccination strategies economically viable given the large number of imported animals and the frequency of the infection? a clear policy in which full virus genome sequences are generated from every outbreak in the country and in which virus from subsets of imported camels is routinely screened and sequenced after 2 years, would provide incredibly useful information about the transmission patterns of the virus and how to stop it. certainly the resources and expertise to perform this sequence monitoring are available and only governmental support is needed to run such a survey. the cost of such a survey would be far less than the management costs and grief associated with a single hospital outbreak. numerous priority research questions regarding mers-cov (basic science, epidemiology, management, and development of new diagnostics, biomarkers, treatments, and vaccines) in both humans and camels, highlighted 2 years ago by the who mers expert groups 35 and by others, 36 remain unanswered. these have again been raised recently, highlighted by calls from saudi arabian health care staff and scientists 37, 38 and by yet another who mers expert group, which has defined a ''roadmap for research and product development against mers-cov''. 39 in 2000 the who set up the global outbreak alert and response network (goarn) 40 for better coordination of surveillance efforts across the globe. it networks 150 institutions and partner agencies, with cooperation with other agencies such as public health england and the us centers for disease control and prevention (cdc) and consortia such as the international severe acute respiratory and emerging infection consortium (isaric). 41 recent consortia such as glopid-r aim to bring together research funding organizations on a global scale to facilitate an effective research response within 48 h of a significant outbreak of a new or re-emerging infectious disease with pandemic potential. 42 the past 4 years has seen outbreaks of ebola virus, zkv, and mers-cov, [2] [3] [4] 13 which indicate that the global community needs to seriously reflect on what is critically missing from current political, scientific, and public health agendas, and how to delineate what is required at the national, regional, and global levels to prevent future epidemics. the factors and operating conditions that promote the emergence and geographical spread of zoonoses are complex and may be related to a single event or chain of multiple events influenced by the genetic evolution of the pathogen, environmental and climate changes, anthropological and demographic changes, and movement and behaviour of humans, animals, and vectors. with animal, human, and environmental factors playing a critical role in its evolution, mers-cov requires more close collaboration between human and animal health systems and university academics to reduce the risk of pandemic spread. 43 moreover, a better understanding of the agricultural dynamics involved in its persistence and spread in camels and studies on interactions between hosts in the environment are urgently needed. the intermittent detection and reporting of mers cases in the community and sporadic nosocomial mers-cov outbreaks will require a more coordinated response plan to study clinical cases, conduct translational basic science and clinical trials research, and perform longitudinal sequencing studies from human and camel mers-cov isolates. a more collaborative mers-cov response plan is required to better define mers-cov epidemiology, transmission dynamics, molecular evolution, laboratory capacity, optimal treatment and prevention measures, and development of vaccines for humans and camels. 44 a better understanding of the prevailing disease ecology and investigations into the dynamics of infectious agents in wildlife could act as a better means of preventing outbreaks in livestock and people at source. the 'one health' concept is an important concept that focuses on the relationship and interconnections between humans, animals, and the environment, and recognizes that the health and wellbeing of humans is intimately linked to the health of animals and their environment (and vice versa). [45] [46] [47] [48] [49] a balanced ecological approach improves understanding of the true threat of novel pathogens and helps to avoid costly, poor, and inappropriate responses to new diseases. in many cases, solutions can be found through altered development pathways and are not inevitably requiring of costly, unsustainable technical and pharmaceutical interventions. thus it is ideally suited to the mers-cov situation in which camels, humans, and environmental factors are central to its persistence and evolution. since the kingdom of saudi arabia is host to millions of pilgrims each year travelling from all continents, 29 tackling the threat of mers and other infectious diseases with epidemic potential will require enhanced closer cooperation between those who provide human health, animal health, and environmental health services, locally, nationally, regionally, and internationally: the middle eastern, european, african, asian, and american governments, veterinary groups, the who, the food and agriculture organization (fao), the african union, the united nations international children's emergency fund (unicef), the world bank, office international des epizooties (oie), cdc, public health england, the newly formed africa cdc, and funding agencies among others. they should now demonstrate increased commitment towards local, national, and global multidisciplinary collaborative efforts to secure optimal health for people, animals, and the environment. global efforts need to be focused on establishing the capability for and strengthening of surveillance systems in developing countries, particularly in africa where emerging and re-emerging zoonoses are a recurrent problem. a prime emphasis should be on developing awareness and response capacity in all countries and on promoting interdisciplinary collaboration and coordination. critical to the establishment of a well-functioning 'one health' platform is the creation of a multidisciplinary team with a range of expertise, including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral geneticists, and researchers, with easy access to adequate laboratory facilities, who will collaborate in order to learn more about zoonotic spread between animals, humans, and the environment and to monitor, respond to, and prevent major outbreaks. there is an urgent and critical need to build a sustainable public health programme and rapid response capability for outbreaks of zoonotic pathogens in the middle east and in low-income countries, especially in africa. importantly there is a need for capacity development programmes designed to strengthen research training and build career pathways for the best and brightest post-doctoral researchers, including phd and masters students working at the interface of humans, animals, and environment. these should include national or regional laboratory facilities, as surveillance requires laboratory support to be meaningful. the development of human and animal health research leaders will create a critical mass of local research capacity and the development of self-funding research environments in african universities and research institutes. this capacity growth could be facilitated through the further development and support of a geographical network of equitable and enduring south-south and north-south partnerships. 9. need for more effective political and scientific engagement to eradicate the threat of mers-cov and other zoonotic diseases the persistence of mers-cov 4 years since its first discovery has created major opportunities for each of the middle eastern and african countries to take leadership of the 'one health' approach with a view to bringing this under regional and global umbrellas, to tackle new emerging and re-emerging infectious diseases with epidemic potential. this will also devolve current dominance of the global health agenda by western groups and consortia and allow equitable partnerships to be established with long-term sustainability. the past year has seen some progress in research into mers-cov, but there remains a need for a more effective, coordinated, and multidisciplinary 'one health' consortium to take forward mers-cov research on priority areas already defined by saudi scientists 37, 38 and the who mers committee. 39 the establishment of regional 'one health' centres of excellence in the middle east (under the league of arab states) and at specific geographical locations in west, central, east, and southern africa could make an important difference in mitigating the risks and factors that pose a risk to both human and animal health. furthermore, any operational plan developed will contribute to strengthening the sentinel surveillance systems in sub-saharan africa in the preparedness and response to potential outbreaks. regional centres should be sufficiently empowered to manage the spectrum of 'one health' approaches to zoonotic disease control in humans and animals, from behaviour change and social interventions for prevention to surveillance of infections and antimicrobial resistance, and preparedness and response to outbreaks. a model for the major syndromes (respiratory, neurological, haemorrhagic, gastro-enteric, and sepsis-like presentations) should be developed so that clinical protocols may be adapted rapidly for any major outbreak during mass gatherings. this should include the development and introduction of innovative and smart platforms for data sourcing, sample collection, and analysis, in order to give clinicians and public health workers continuously updated information on which clinical decisions may be based. there is a pressing need to develop and strengthen the national ethics and medicines regulatory frameworks in sub-saharan africa in order to strike a balance between the public health interest, the interests of the pharmaceutical industry, and ethical values. parallel initiatives across africa and the tropics could be harmonized to create regional networks that can serve as a repository for expert 'one health' advice on agriculture, sustainable livestock, and the links to human development. there are several ongoing important initiatives on developing 'rapid response' and broader 'one health' capacity development groups in europe, asia, and the americas to assist in the surveillance and response to emerging infectious disease threats. the public health systems of west african countries failed with the ebola epidemic, and the response from the who and the international community was very slow and uncoordinated. this led to thousands of people, including over 500 health care workers, losing their lives. the factors governing the appearance and disappearance of new coronaviruses affecting humans are complex and it has been over 4 years since the first patient died of mers-cov. mers cases continue to be reported throughout the year from the middle east. there is a large mers-cov camel reservoir and there is no specific treatment or vaccine. the precise pathway from infected camel to the recurring mers hospital outbreaks needs to be understood in order to devise effective control measures. with 10 million people visiting saudi arabia every year for umrah and/or hajj and the increasing importation of live animals from sub-saharan africa, the potential risk of global spread will be everpresent, especially if mutations or recombinations in mers-cov occur. a major 'one health' initiative to tackle mers-cov at source in animal populations is thus required. 50 middle eastern and african governments should now work more closely together and increase collaborative efforts with international partners and global public health authorities if we are to prevent yet another global zoonotic pandemic. conflict of interest: all authors have a specific interest in 'one health'. the authors declare no conflicts of interest. there was no financial support. emerging and re-emerging infectious threats in the 21st century world health organization. ebola virus disease outbreak world health organization. zika virus be prepared: europe needs ebola outbreak consortium ethics for pandemics beyond influenza: ebola, drug-resistant tuberculosis, and anticipating future ethical challenges in pandemic preparedness and response ebola: missed opportunities for europe-africa research rapid spread of zika virus in the americas-implications for public health preparedness for mass gatherings at the 2016 brazil olympic games lessons from the ebola outbreak: action items for emerging infectious disease preparedness and response challenges in controlling the ebola outbreak in two prefectures in guinea: why did communities continue to resist? world health organization. who publishes list of top emerging diseases likely to cause major epidemics world health organization. a research and development blueprint for action to prevent epidemics 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coordinating global activities to address health risks at the animal-human-ecosystems interfaces. a tripartite concept note international organization for standardization. who develops iso standards. geneva: who towards a one health approach to controlling zoonotic diseases key: cord-303272-1w8epdht authors: reusken, chantal bem; haagmans, bart l; müller, marcel a; gutierrez, carlos; godeke, gert-jan; meyer, benjamin; muth, doreen; raj, v stalin; vries, laura smits-de; corman, victor m; drexler, jan-felix; smits, saskia l; el tahir, yasmin e; de sousa, rita; van beek, janko; nowotny, norbert; van maanen, kees; hidalgo-hermoso, ezequiel; bosch, berend-jan; rottier, peter; osterhaus, albert; gortázar-schmidt, christian; drosten, christian; koopmans, marion pg title: middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study date: 2013-08-09 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70164-6 sha: doc_id: 303272 cord_uid: 1w8epdht background: a new betacoronavirus—middle east respiratory syndrome coronavirus (mers-cov)—has been identified in patients with severe acute respiratory infection. although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of mers-cov. anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. we investigated possible animal reservoirs of mers-cov by assessing specific serum antibodies in livestock. methods: we took sera from animals in the middle east (oman) and from elsewhere (spain, netherlands, chile). cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum igg by protein microarray using the receptor-binding s1 subunits of spike proteins of mers-cov, severe acute respiratory syndrome coronavirus, and human coronavirus oc43. results were confirmed by virus neutralisation tests for mers-cov and bovine coronavirus. findings: 50 of 50 (100%) sera from omani camels and 15 of 105 (14%) from spanish camels had protein-specific antibodies against mers-cov spike. sera from european sheep, goats, cattle, and other camelids had no such antibodies. mers-cov neutralising antibody titres varied between 1/320 and 1/2560 for the omani camel sera and between 1/20 and 1/320 for the spanish camel sera. there was no evidence for cross-neutralisation by bovine coronavirus antibodies. interpretation: mers-cov or a related virus has infected camel populations. both titres and seroprevalences in sera from different locations in oman suggest widespread infection. funding: european union, european centre for disease prevention and control, deutsche forschungsgemeinschaft. in 2012, a new betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-was identifi ed in patients with severe respiratory disease in the middle east. as of aug 2, 2013, 94 laboratory-confi rmed cases, including 46 deaths, have been reported to who. 1 illness associated with mers-cov infection is characterised primarily by mild-to-severe respiratory complaints, most requiring hospital admission for acute respiratory distress syndrome. comorbidities and immunosuppression seem to predispose for infection and severe disease, [2] [3] [4] [5] [6] and unpublished serological studies suggest that asymptomatic infections occur. 7 all cases reported so far have been linked to jordan, qatar, saudi arabia, and united arab emirates. humanto-human transmission has been reported, particularly in health-care settings, but on the basis of available evidence the basic reproduction number (r 0 ) is thought to be low, suggesting that the virus is not transmitted readily. 6, 8 therefore, the primary reservoir of mers-cov is probably animals. diff erent coronaviruses have various hosts including wildlife, livestock, poultry, pets, and human beings. coronaviruses can adapt to new host species, as shown by the zoonotic origin of several human coronaviruses. 9 human coronavirus oc43 has recent common ancestry with bovine coronaviruses. 10 rhinolophid bats were identifi ed as a likely reservoir for severe acute respiratory syndrome coronavirus (sars-cov), which emerged in people in 2002-03, through intermediate carnivorous hosts. 11 molecular clock analysis 12 showed that bat and civet strains of viruses closely related to sars-cov only diverged a few years before the outbreak. human coronavirus 229e has a common ancestor with coronaviruses found in ghanaian hipposideros spp bats. 13 mers-cov is able to replicate in various bat cell lines 14 and phylogenetic analyses show that it is closely related to betacoronavirus lineage c viruses from pipistrellus spp bats in europe and asia. [15] [16] [17] [18] molecular clock dating of epidemiologically unlinked isolates of human mers-cov estimated their divergence from a common ancestor in mid-2011, 4, 19 with a cluster of isolates from the eastern arabian peninsula diverging in late 2012. 4 this fi nding could suggest that the diversity of mers-cov in people is the result of multiple independent, geographically structured, zoonotic events in the middle east. 4, 19 possible animal reservoirs need to be identifi ed to determine how circulation of mers-cov is maintained and to break the chain of transmission. 20 mers-cov can infect cells of several species, including human beings and bats. 14 the functional receptor is conserved between species, suggesting that receptor use is not an important barrier to cross-species transmission. 21 data for exposure history of patients are scarce, but suggest contact with livestock, including dromedary camels and goats. 2, 4, 5 food and agriculture organization data from 2011 show that cows, goats, sheep, and dromedary camels are the main sources of meat and milk in jordan, saudi arabia, and united arab emirates. 22 serological studies are best suited to screen animal populations, but have not yet been reported for mers-cov in animals, although several methods have been described for testing antibodies of people. 23, 24 for specifi city, who recommends use of a combination of screening assays with recombinant spike protein, and confi rmatory testing by neutralisation assays. here, we describe antibody profi ling of serum samples from major livestock species that might be relevant to the epidemiology of mers-cov in the middle east, using samples collected from herds inside and outside the region. we sampled a cohort of 105 dromedary camels (camelus dromedarius) from two herds on the canary islands. 50 were male, 55 were female, 88 were adults, nine were age 3-4 years, seven were age 2 years, and one was age 3 months. both herds had the same owner, with frequent exchange of animals between the herds. one herd is from a coastal dune habitat with no other livestock, while the other herd is in an inland valley close to a tropical fruit farm, in particular mango and papaya-which could attract fruit bats-and nearby (roughly 500 m) to horse and goat farms with 25 and 300 animals, respectively. the camels were born in the canary islands except for three adults, which were imported from morocco. 25 the camels are used in the tourist industry. 80 sera were taken april-june, 2012, nine in may, 2013, and 16 paired sera were taken in these months in 2012, and 2013, all for routine veterinary purposes. samples were obtained by jugular puncture. 50 female dromedary camels from oman were sampled in march, 2013. the camels were aged 8-12 years and belonged to diff erent owners from separate locations. the camels are retired racing camels now used for breeding, and blood was taken by jugular puncture for routine screening for brucellosis. omani dromedaries sera were collected for veterinary purposes from two llamas (lama glama), six alpacas (vicugna pacos), and two bactrian camels (camelus bactrianus) in the netherlands. sera were collected for veterinary purposes from two bactrian camels, 18 alpacas, fi ve llamas, and two guanaco (lama guanicoe) in buin zoo in chile. sera from cattle (n=40), domestic goats (n=40), and sheep (n=40) were from routine submission to the dutch animal health service. sera from spanish domestic goats (n=40) were provided by the instituto de investigación en recursos cinegéticos (ciudad real, spain) from submissions for tuberculosis control in 2011. all sera were obtained in agreement with local regulations and dutch import regulations with regard to animal disease legislation. positive human control sera for the three antigens used on the microarray were taken as described previously. 24 all samples were stored at -20°c until testing. we tested the sera for the presence of igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc43 s1 antigens in a protein microarray. the receptor-binding domains, which contain the s1 subunit of spike proteins of mers-cov (residues 1-747), sars-cov (residues 1-676), and human coronavirus oc43 (residues 1-760) were expressed, purifi ed, and spotted on glass slides. slides were incubated with serum and species-specifi c conjugates, as previously described. 24 goat sera were incubated with alexa fluor 647-conjugated rabbit anti-goat igg fc fragment (jackson immuno research, west grove, pa, usa); combinations of the mean fl uorescent intensities of reactions of sera with mers-cov and human coronavirus oc43 antigens from 105 spanish dromedary camels (a). plaque reduction neutralisation tests for bovine coronavirus and mers-cov (b): two representative sera are shown (numbers 15 and 5, corresponding to camel id numbers in table 2) in dilutions of 1/40, 1/160, and 1/640 as well as the virus input control. all samples were tested in duplicates (only one well shown) and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90%. igg reactivity of both camel sera to mers-cov antigen and human coronavirus oc43 antigen in a two-step dilution series in the microarray (c). igg reactivity of two two-step serially diluted omani dromedary camel sera with human coronavirus emc antigen and human coronavirus oc43 antigen in the microarray (d). rfu=relative fl uorescence units. mers-cov=middle east respiratory coronavirus. cow sera with alexa fluor 647-conjugated goat antibovine igg (h+l; jackson immuno research); sheep sera with alexa fluor 647-conjugated donkey anti-sheep igg (h+l; millipore, temecula, ca, usa); and camelid sera with dylight 650-conjugated goat anti-llama igg (h+l; agrisera, vännas, sweden). fluorescence signals were quantifi ed as described previously. 24 we tested the sera for igg reactivity at a dilution of 1/20 and set an arbitrary cutoff at an average signal intensity of 20 000 relative fl uorescence units (rfu). this high cutoff was chosen to reduce cross-reactive false positives. 24 we present results as rfu for each set of quadruplicate spots per antigen. negative fl uorescent intensities (caused by background correction) were assigned to 0. analyses were done with graphpad prism (version 6.02). sera were heat-inactivated before virus neutralisation by incubation for 30 min at 56°c. two-fold serial dilutions of sera were prepared using 96-well plates, starting dilution 1/10. mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with clemizole penicillin (penicillin g), streptomycin, and 1% fetal bovine serum, to a dilution of 2000 tissue culture infective dose 50 per ml. 50 μl virus suspension was added to the plates and the plates were incubated at 37°c for 1 h. the mixtures of virus and serum were then incubated on 96-well plates containing vero cells for 1 h followed by washing with phosphate buff ered saline and incubation with imdm and 1% fetal bovine serum for 3-4 days, after which endpoint titres were measured. all tests were repeated twice independently. we tested neutralisation activity of sera against mers-cov (erasmus mc isolate) and bovine coronavirus (nebraska strain) by plaque reduction neutralisation test (90% plaque reduction) with african green monkey kidney cells (cell line vero b4; dsmz acc 33) or bovine kidney cells (cell line pt; cclv-rie11) in a 24-well plate format. virus (30-60 plaqueforming units) and heat-inactivated sera (diluted from 1/40 to 1/640) were pre-incubated in 200 μl of serumfree optipro medium (life technologies, karlsruhe, germany) at 37°c for 1 h. virus adsorption was done at 37°c for 1 h. supernatants were removed and overlaid with avicel resin (fcm biopolymer, brussels, belgium). 5 assays were stopped after 3 days by fi xation with 8% paraformaldehyde for 30 min. all samples were tested in duplicate and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90%. the sponsors had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. sera were tested for igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc43 s1 antigens in a protein microarray (fi gure 1). human coronavirus oc43 is serologically closely related to bovine coronavirus, 26 diverging at the end of the 19th century. 10 bovine coronavirus circulates in cows, sheep, goats, and old and new world camelids. [27] [28] [29] [30] because bovine coronavirus s1 was not available, human coronavirus oc43 s1 antigen was used as a proxy. sera from three llamas, four alpacas, one guanaco, and two bactrian camels reacted with human coronavirus oc43 antigen. one cow and one goat serum reacted with human coronavirus oc43 antigen as did sera from 16 of 105 (15%) spanish dromedary camels. all sera from cattle, sheep, and goats tested negative for mers-cov antigen, but sera from 15 spanish camels (14%) did react with mers-cov antigen (fi gure 1). the reactivity was highly specifi c-the same sera did not bind to sars-cov antigen but a positive control specimen did. no correlation existed between the reactivity of sera with mers-cov antigen and human coronavirus oc43 antigen (fi gure 2). all but one serum sample that reacted with mers-cov antigen were from adult animals. one reactive serum was from a 2-year-old animal. to confi rm the presence of mers-cov specifi c igg in the spanish camel sera, we used a mers-cov neutralisation assay to test a subset of 49 camel sera with diff erent degrees of reactivity with mers-cov and human coronavirus oc43 antigen according to microarray. nine spanish camels had mers-cov neutralising antibodies with titres varying between 1/20 and 1/320 (table 1). three of the 12 sera reacted with mers-cov spike antigen but did not neutralise mers-cov, most likely because of recognition of nonneutralising epitopes. all mers-cov neutralising sera had (almost) saturating reactivity with mers-cov antigen on the microarray, whereas reactivity with human coronavirus oc43 antigen varied from negative to 50% of saturating reactivity (table 1). the variable human coronavirus oc43 signals suggest that mers-cov did not generally cross-react with human coronavirus oc43 or bovine coronavirus antigens. all nine camels with mers-cov neutralising antibodies were born and raised on the canary islands; seven were female, two were male. eight camels were adults, one was 2 years old. to show that the reactivity of the camel sera with human coronavirus oc43 antigen according to the microarray was caused by the presence of bovine coronavirus igg and to further exclude mers-cov neutralising activity caused by cross-neutralisation by the bovine coronavirus antibodies, we tested camels that had suffi cient serum left (n=15) in a comparative mers-cov and bovine coronavirus plaque reduction neutralisation test (fi gure 2, table 2). all camel sera neutralised bovine coronavirus, but with varying titres, suggesting a lower cutoff than 20 000 rfu for oc43 in the microarray (fi gure 1). five camels had high neutralising antibody titres against bovine coronavirus (and a mean signal intensity of greater than 50 000 rfu for human coronavirus oc43 antigen on microarray) but were negative for mers-cov neutralisation, suggesting that cross-neutralisation in this direction did not occur and that the mers-cov neutralising activity was not caused by the presence of bovine coronavirus neutralising antibodies. a serum sample from a patient who had mers, neutralised mers-cov with a high titre (1/640) but neutralised bovine coronavirus less effi ciently (titre 1/80). the latter fi nding was most probably caused by previous infection with human coronavirus oc43-this patient had a high titre (1/>5120) in a human coronavirus oc43 recombinant spike immuno fl uorescence assay and a saturating signal with human coronavirus oc43 antigen in the microarray. two human serum samples positive for human coronavirus oc43 did not neutralise mers-cov, one of which neutralised bovine coronavirus at a titre of 1/80 (table 2) . we tested 50 sera from dromedary camels in oman at a dilution of 1/20 by microarray and mers-cov neutralisation test. all the sera showed saturating reactivity with mers-cov antigen on the microarray, no sars-cov antigen reactivity, and human coronavirus oc43 antigen reactivity varying between negative (below the cutoff of 20 000 rfu) and saturating signals (fi gure 1, table 1). serial dilution of two sera with saturating reactivity for both antigens at the initial dilution of 1/20 showed that mers-cov antigen reactivity was still above the cutoff at 1/5120, whereas human coronavirus oc43 antigen reactivity fell below the cutoff at dilutions of 1/80 to 1/320 (fi gure 2d). consistent with the microarray data, all sera had high mers-cov neutralising capacity, with titres varying between 1/320 (seven of 50 samples) and 1/2560 or more (16 or 50 samples). in this study we describe the presence of mers-cov neutralising antibodies in dromedary camels both in a mers-cov linked (oman) and unlinked regions (canary islands). all the sera from dromedary camels from oman and some from spain had specifi c igg reactivity with the mers-cov receptor binding domain s1. we confi rmed our expectation that another betacoronavirus-bovine coronavirus-circulated in these camelids. 29 spanish camels (9%) had specifi c neutralising antibodies against mers-cov that were clearly not caused by cross-neutralisation by bovine coronavirus antibodies. our study is the fi rst in which animals have been tested for the presence of antibodies specifi c to mers-cov (panel). animal screening is necessary to understand the epidemiology of mers-cov. at present, bats are thought to be the ultimate reservoirs for several established human coronaviruses as well as sars-cov. accordingly, phylogenetic analysis has shown that mers-cov is related to betacoronavirus lineage c viruses found in pipistrellus spp bats. 15, 16 however, direct transmission of mers-cov to people from bats seems unlikely. 4, 19 the identifi cation of possible intermediate hosts that are probably in closer contact with people (eg, livestock) is urgently needed. common livestock species in the middle east include dromedary camels but also cattle, sheep, and goats. based on the available data, we cannot rule out circulation of a mers-related coronavirus in these species-sera were not available from epi demi ologically linked regions. the high prevalence of mers-cov neutralising antibodies in dromedary camels from oman suggests circulation of mers-cov or a closely related virus in this population. however, attempts to identify viral sequences in spanish camel sera and faecal samples using pancoronavirus and specifi c betacoronavirus 2c pcr methods 15, 31, 32 were unsuccessful (unpublished data), as was untargeted amplifi cation followed by deep sequencing of faecal samples (unpublished data). these results imply that the camels were not actively shedding the virus at the time of sampling. less than 10% of the animals in the canary islands had mers-cov neutralising sera with titres up to 1/320. this low seroprevalence means either that exposure of the animals to other putative reservoirs is rare 33 or that the virus is absent in this closed-off population of roughly 2000 animals. 25 we cannot rule out that the population might have once had an outbreak but that by the time of sampling, antibody titres had waned and no new introductions of the virus had occurred. the camels have contact with wild rodents, rabbits, pigeons, and doves and possibly also with bats. seven insectivorous bat species, including three pipistrellus spp, are native to the canary islands, while egyptian fruit bats (rousettus aegyptiacus) have been introduced. 34 the 100% seroprevalence with high titres in omani camels from diff erent owners and locations suggests a diff erent situation in the middle east, with widespread circulation of mers-cov or a closely related virus. this diff erence of epidemiology might be because the virus circulating in the middle east is diff erent to that circulating in spain, with increased animal transmissibility and human infections. 35 in addition, the omani camels were once racing camels now held for breeding and might be kept in circumstances that favour virus transmission. for cattle, a relation has been established between the incidence and eff ects of respiratory diseases, management practices, and animal transport. 36, 37 to our knowledge, the camel populations in oman and the canary islands are not connected. camels on the canary islands were originally imported in the 15th century from the horn of africa for labour and transport. nowadays, import of animals from africa is banned because of the risk of foot-and-mouth disease. only three camels in our study were originally imported from morocco, more than 18 years ago. because the closest relatives of mers-cov were identifi ed very recently in neoromicia zuluensis bats from africa, 38 the introduction of mers-cov or related viruses into some african camel populations could have occurred decades ago, giving a possible explanation for mers-cov antibodies in camels from the canary islands. in the middle east, huge numbers of camels are imported from africa to meet the demand for meat. the top fi ve camel breeding countries are all african, and saudi arabia and united arab emirates are in the top fi ve camel meat producing countries. 22 this increased turnover of animals in the middle east we searched pubmed for "novel coronavirus emc" or "mers-cov", we identifi ed 43 reports in english linked to the middle east respiratory syndrome coronavirus (mers-cov) published before july 22, 2013. none of these reports described a serological study for mers-cov-specifi c antibodies in animals. our report describes the fi rst mers-cov serological study of major livestock relevant to the middle east. our study shows that mers-cov or a related virus has infected dromedary camel populations. both titre levels and seroprevalences in sera from diff erent locations in oman suggest widespread infection of camelids with mers-cov or a closely related virus. targeted studies are needed to confi rm our fi ndings and their possible relevance to human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels from diff erent regions for which epidemiological background information is available, as well as virological assessment of samples from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime, we recommend a detailed case history of confi rmed mers-cov cases, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. compared with the canary islands could also aff ect the epidemiology of a virus, through more frequent infl ux of immunologically naive animals. targeted studies are needed to confi rm our fi ndings and their possible relevance in relation to the human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels for which epidemiological background information is available, as well as virological assess ment of specimens from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime we recommend a detailed case history of people with mers-cov, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. who. global alert and response (gar): novel coronavirus infection recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov)-update interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk coronavirus diversity, phylogeny and 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relationships between human coronavirus oc43 and neonatal calf diarrhoea coronavirus serum antibodies to bovine coronavirus in swedish sheep serosurveillance of viral diseases in korean native goats (capra hircus) enteric coronavirus infection in a juvenile dromedary analysis of the genome sequence of an alpaca coronavirus the diff erential clinical impact of human coronavirus species in children with cystic fi brosis generic detection of coronaviruses and diff erentiation at the prototype strain level by reverse transcription-pcr and nonfl uorescent low-density microarray forecast and control of epidemics in a globalized world la fauna de quiropteros del archipelgo canario coronavirus genomics and bioinformatics analysis risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds associations between the distance traveled from sale barns to commercial feedlots in the united states and overall performance, risk of respiratory disease, and cumulative mortality in feeder cattle during 1997 to 2009 close relative of human middle east respiratory syndrome coronavirus in bat we thank prof mc horzinek for helpful suggestions. rds was funded by the european public health training program (euphem), ecdc, stockholm, sweden. contributions to the study were funded through the european union fp7 projects emperie (contract number 223498; to blh, sls, ao, cd) and antigone (contract number 278976; to cg, cd, mpgk, ao). work in bonn was also funded by deutsche forschungsgemeinschaft (dfg grant dr772/3-1 to cd). key: cord-354302-l2kywzro authors: adney, danielle r.; van doremalen, neeltje; brown, vienna r.; bushmaker, trenton; scott, dana; de wit, emmie; bowen, richard a.; munster, vincent j. title: replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels date: 2014-12-17 journal: emerg infect dis doi: 10.3201/eid2012.141280 sha: doc_id: 354302 cord_uid: l2kywzro in 2012, a novel coronavirus associated with severe respiratory disease in humans emerged in the middle east. epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of middle east respiratory syndrome coronavirus (mers-cov). here we provide experimental support for camels as a reservoir for mers-cov. we inoculated 3 adult camels with a human isolate of mers-cov and a transient, primarily upper respiratory tract infection developed in each of the 3 animals. clinical signs of the mers-cov infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. we detected infectious virus in nasal secretions through 7 days postinoculation, and viral rna up to 35 days postinoculation. the pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-to-human transmission. in 2012, a novel coronavirus associated with severe respiratory disease in humans emerged in the middle east. epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of middle east respiratory syndrome coronavirus (mers-cov). here we provide experimental support for camels as a reservoir for mers-cov. we inoculated 3 adult camels with a human isolate of mers-cov and a transient, primarily upper respiratory tract infection developed in each of the 3 animals. clinical signs of the mers-cov infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. we detected infectious virus in nasal secretions through 7 days postinoculation, and viral rna up to 35 days postinoculation. the pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-tohuman transmission. t he middle east respiratory syndrome coronavirus (mers-cov) was first recognized in 2012 related to a fatal human case of pneumonia in saudi arabia (1) . currently, >800 cases of mers have been identified, and the estimated case-fatality rate is ≈35% (2) . most cases have been identified on the arabian peninsula, but several travel-associated cases have been reported (2) (3) (4) . human-to-human transmission has been reported, predominantly among persons in health care facilities and households; the rate of human infection by zoonotic transmission from a reservoir source is currently not known (4) (5) (6) . the close phylogenetic relationship of human mers-cov isolates with those obtained from bats initially suggested a direct link between the emergence of mers-cov and a putative natural reservoir (7) (8) (9) . anecdotal reports mentioned contact of mers-cov-infected patients with camels and goats, suggesting that livestock might be the intermediate reservoir host for mers-cov (4,10-12). serologic studies revealed widespread prevalence of mers-cov-specific antibodies in dromedary camels from several countries that reported mers cases (4, (13) (14) (15) (16) (17) (18) (19) . further, mers-cov rna was detected in nasal swab samples obtained from 3 camels on a farm linked to 2 human mers-cov cases, and the virus was isolated from nasal swab samples from dromedary camels in qatar (14) . mers-cov isolation and subsequent full genome sequencing directly linked a dromedary camel and a fatal mers-cov case in a person in saudi arabia (20, 21) . despite these associations, the role of camels as a primary reservoir for mers-cov is still debated (22, 23) . here we report on the experimental inoculation of 3 camels with a human isolate of mers-cov. mers-cov (strain hcov-emc/2012) was provided by the department of viroscience, erasmus medical center, rotterdam, the netherlands. the virus was propagated in vero e6 cells cultured in dulbecco modified eagle medium (invitrogen, carlsbad, ca, usa) supplemented with 2% fetal bovine serum, 2 mmol/l glutamine, 50 u/ml penicillin, and 50 µg/ml streptomycin. three native-born adult male dromedary camels (camelus dromedarius) were obtained through private sale; the animals tested negative by neutralization assay for mers-cov and for bovine coronavirus by elisa. camels 1, 2, and 3 were 2, 3, and 5 years old, respectively. camels 1 and 2 were intact males, and camel 3 had been castrated. animals were housed in an animal biosafety level 3 facility for the duration of the experiment and fed ad libitum. camels were acclimated to the facility for 2 weeks before virus inoculation. we sedated the camels with xylazine, then inoculated them with a total dose of 10 7 50% tissue culture infective dose (tcid 50 ) of mers-cov (strain hcov-emc/2012) in a total volume of 15 ml, by way of intratracheal (8 ml using transcutaneous catheter), intranasal (3.3 ml in each nostril by expulsion from a syringe), and conjunctival (0.2 ml in each conjunctival sac) routes. the routes of inoculation and infectious dose were chosen to reflect a combination of most likely routes of exposure and to increase the potential of infection. the animals were observed at least 1× daily for the duration of the experiment for behavior, food consumption, activity level, and nasal discharge. rectal temperature was taken daily from 2 to 7 days postinoculation, then 3× weekly until the animals were euthanized. nasal and oral swab samples and fecal samples were collected into virus transport medium or virus lysis buffer daily from 0 to 7 days postinoculation (dpi), then 3× weekly until the animal was euthanized. blood was collected into evacuated edta and serum-separating tubes daily at 0-7 dpi and 3× weekly thereafter. urine was collected by convenience and at necropsy. to evaluate whether virus is exhaled from infected camels, a funnel was placed over the muzzle of each camel and connected to a vacuum pump to capture exhaled air in tissue culture media (10 ml dulbecco modified eagle medium, 1% fetal bovine serum, 0.013% se-15 (anti-foam) with an all glass impinger (ace glass inc., vineland, nj, usa). exhaled breath was collected for ≈2 minutes and analyzed by quantitative real-time pcr (qpcr) and virus titration. on days 5, 28, and 42, camels 1, 2, and 3, respectively, were euthanized, and samples were collected from nasal turbinates, lungs, trachea, larynx, pharynx, liver, spleen, kidney, bladder, urine, duodenum, jejunum, colon, rectum, abomasum, forestomachs, prescapular lymph node, retropharyngeal lymph node, tracheobronchial lymph node, mediastinal lymph node, mesenteric lymph node, medulla, and olfactory cortex. we extracted rna from swab samples, fecal samples, and serum samples using the qiaamp viral rna kit (qia-gen, valencia, ca, usa) according to the manufacturer's instructions. for detection of viral rna, we used 5 ml of rna in a one-step real-time reverse transcription pcr upe assay (24) using the rotor-genetm probe kit (qiagen) according to manufacturer's instructions. standard dilutions of a titered virus stock were run in parallel, to calculate tcid 50 equivalents in the samples (25) . we titrated swab samples in viral transport medium, whole blood, and homogenized tissues (≈10% wt/vol) for mers-cov virus by plaque assay. briefly, 10-fold serial dilutions of samples were prepared in ba-1 medium (mem, 1% bovine serum albumin, 350 mg/l sodium bicarbonate, 50 mm tris, ph 7.6, 5 mg/l phenol red) containing 100 mg gentamicin, 200,000 u penicillin g, 100 mg streptomycin, and 5 mg amphotericin/l; plaque assay was conducted as horizontal lines indicate the normal temperature range observed among these dromedary camels as calculated by mean ± 3×, the sd before inoculation. described for west nile virus (26) . plaques were counted on days 1 and 3 after the second overlay and virus titers were expressed as pfus per ml or gram. we determined neutralizing antibody titers by plaque reduction neutralization test as described (26), using a 90% neutralization cutoff. tissues were fixed for >7 days in 10% neutral-buffered formalin and embedded in paraffin. tissue sections were stained with hematoxylin and eosin. to detect mers-cov antigen, we completed immunohistochemical testing using a rabbit polyclonal antiserum against hcov-emc/2012 (1:1,000) as a primary antibody. each camel showed minor clinical signs of disease, consisting of rhinorrhea (figure 1, panel a) and a mild elevation in body temperature at 2 dpi and 5-6 dpi ( figure 1, panel b) ; no other clinical signs were observed. rhinorrhea developed in all 3 camels beginning at 2 (camels 1 and 3) and 5 (camel 2) dpi, and persisted <2 weeks. the nasal discharge drained from both nares and varied in character from serous to purulent; minor hemorrhage was observed on some occasions, but may have been caused by trauma that occurred during collection of samples. mers-cov shedding started during 1-2 dpi, as detected by the presence of infectious virus and viral rna by qpcr in nasal swab samples. infectious virus shedding was detected <7 dpi, and shedding of viral rna was detected <35 dpi in nasal swab samples (figure 2 ). low concentrations of infectious virus and viral rna were detected in oral samples, likely originating in drainage from the nasal cavity ( figure 3) . no viral rna was detected in fecal samples or in urine samples collected by convenience or at necropsy at 0, 1, 5, 14, 21, 28, and 42 dpi from the 3 camels. no infectious virus or viral rna was detected in any of the serum or whole blood samples. small quantities of mers-cov rna were detected in exhaled breath by qpcr (10 1.2 and 10 1.4 tcid 50 equivalent/ml) at 3 and 5 dpi, but infectious virus was not detected. infectious virus was detected in tissues from camel 1, which was euthanized on 5 dpi, but not in tissues obtained from camels 2 and 3, which were euthanized at28 and 42 dpi, respectively. infectious virus was detected in tissues of the upper respiratory tract (urt), including nasal turbinates, olfactory epithelium, pharynx, and larynx. in the lower respiratory tract, infectious virus was detected in the trachea and in 1 of 4 lung lobes tested. infectious virus was also detected in the retropharyngeal, mediastinal, mesenteric, and tracheobronchial lymph nodes (figure 4 ). on necropsy of camel 1 at 5 dpi, histologic lesions were found in the pseudostratified epithelial cells in the urt and the lower respiratory tract (trachea, bronchi, and bronchioles) but not in the alveoli ( figure 5) . the lesions were characterized as mild to moderate acute intraepithelial and submucosal inflammation with multifocal necrosis and loss of pseudostratified epithelial cells, comparable to the common cold among humans. multifocal loss of epithelial polarity and cilia with squamous metaplasia were observed. the epithelium was infiltrated by small-to-moderate numbers of neutrophils with fewer macrophages; similar inflammatory cells also permeated the submucosa. the submucosal glands of the trachea were multifocally necrotic and infiltrated by small numbers of neutrophils. viral antigen was detected within the epithelial cells of the nasal turbinates, larynx, trachea, bronchi, and bronchioles, but not the alveoli. in addition, viral antigen was present at the follicular mantle zone of the tonsils and mediastinal and retropharyncheal lymph nodes ( figure 5 ). the nasal turbinates, larynx, and trachea of camel 2 (necropsied at 28 dpi) had similar but milder lesions when compared with those of camel 1. the nasal turbinate, larynx, and bronchus showed small numbers of infiltrating neutrophils; however, in contrast with the condition of camel 1, the cilia and goblet cells were intact. the remainder of the respiratory tract of camel 2 was unaffected. immunohistochemical testing revealed the presence of limited viral antigen in the nasal turbinate but not in any of the other tissues at that time. no lesions or viral antigens were detected in camel 3 at 42 dpi. serum samples were collected weekly from the camels to monitor the generation of neutralizing antibodies specific to mers cov. each of the 3 camels was seronegative before inoculation. robust mers-cov specific antibody responses developed in camels 2 and 3 (euthanized on 28 and 42 dpi, respectively), detected first on 14 dpi with a plaque-reduction neutralization test titer from 20 to 40 that increased to 640 at 35 dpi (table) . camel 1 was euthanized at 5 dpi and was not tested for development of antibodies against the virus. tissues were collected at 5 days postinoculation (dpi) for camel 1, 28 dpi for vamel 2 and 42 dpi for camel 3. detectable infectious virus in the collected tissues was found only in camel 1. nasal turbinates were sampled in 3 different sections: anterior, medial, and posterior. infectious titers were determined by plaque assay. ln, lymph node. epidemiologic and surveillance data on the emergence of mers-cov strongly point toward a role for dromedary camels as a reservoir for zoonotic transmission (13) (14) (15) (16) (17) (18) (19) (20) (21) 27, 28) . to understand the ecology of mers-cov in the most likely reservoir host, we experimentally inoculated 3 young adult dromedary camels with mers cov. the disease observed was clinically benign, in agreement with the absence of overt illness reported from field surveillance studies (14, 17, 19, 21) . a large quantity of mers-cov and viral rna was detected in nasal swab specimens from each of the 3 camels. infectious virus was detected through 7 dpi, and rna was detected through 35 dpi in camel 3, which was euthanized on day 42. this route of shedding is consistent with data on naturally infected camels (14, 18, 21, 28, 29) , and the pattern of shedding suggests that the infectious period of camels may be short. mers-cov was not detected in either urine or feces, again consistent with field observations (21, 28) . the large quantities of mers-cov shed in nasal secretions by each of the 3 camels suggest that camel-tocamel and camel-to-human transmission may occur readily through direct contact and large droplet, or possibly fomite transmission. histopathologic examination revealed that the urt, specifically the respiratory epithelium in the nasal turbinates, is the predominant site of mers-cov replication in camels. neutralizing antibodies were detected from 14 dpi onward, reaching a maximum neutralizing titer of 640 after 35 days. serologic studies in camels in the field have reported mers-cov neutralizing titers as high as 5,120 (14, 16) , potentially indicative of repeated exposure and re-infection. the study reported here was done on the basis of inoculation of 3 male animals with a human isolate of mers-cov, and the study design we used imposed several limitations on how these data inform what occurs in natural infections. the camels we inoculated were exposed to a high dose of virus by 3 simultaneous routes of inoculation. in retrospect, the inoculation dose does not seem excessive, based on the large quantity of virus shed nasally in all 3 animals ( figure 2 ). the total dose inoculated was relatively equivalent to the amount of virus present in a single nasal swab sample taken during the first days postinoculation, and it seems probable that a camel shedding this quantity of virus would readily infect other camels or humans with which it had direct contact. the fact that we inoculated the camels with the virus by 3 routes precludes drawing conclusions regarding efficiency of transmission by a particular route, which is a topic that should be addressed in future studies. the influence of camel age on susceptibility and dynamics of virus shedding is another notable parameter that requires further study. it seems likely that productive infection and shedding of virus in natural settings occurs predominantly in juvenile camels (28) . this could be the result of an intrinsic difference in age-related susceptibility, but is more likely related to the immunologically naïve status of the animals in the context of a high force of infection after decay of passively acquired antibodies. the animals we infected were young adults, but were seronegative and therefore probably as susceptible as juveniles from mers-cov-endemic regions. another aspect of pathogenesis not addressed here is whether virus is present in milk or meat from infected camels and thereby poses another potential route of exposure to humans who consume such products. despite these limitations, the magnitude and pattern of virus shedding was essentially identical in all 3 animals and supports the available epidemiologic data indicating that camels are likely a major reservoir host for mers-cov. additional experimental and field studies are clearly required to address the duration of shedding of infectious mers-cov from infected camels, to determine whether infection results in protective immunity, and to clarify the burden of illness among humans resulting from transmission from camels. emerging infectious diseases • www.cdc.gov/eid • vol. 20, no. 12, december 2014 isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. global alert and response. middle east respiratory syndrome coronavirus (mers-cov)-update investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in florence the emergence of the middle east respiratory syndrome coronavirus hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus in bats, saudi arabia a decade after sars: strategies for controlling emerging coronaviruses recovery from severe 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evidence for camel-to-human transmission of mers coronavirus concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques dynamics of passive immunity to west nile virus in domestic chickens (gallus gallus domesticus) middle east respiratory syndrome coronavirus antibody reactors among camels in dubai mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through wholegenome analysis and virus cultured from dromedary camels in saudi arabia we thank bart haagmans and ron fouchier, for providing mers-cov (isolate hcov-emc/2012); tina thomas, dan long, and rebecca rosenke for histopathologic examination; and anita mora and ryan kissinger for figure preparation. all animal work in this study was approved by the institutional animal care and use committee of colorado state university and was performed in compliance with recommendations in the guide for the care and use of laboratory animals of the national institute of health.ms adney is a graduate student at colorado state university in fort collins, colorado. her research focus is on the pathogenesis of emerging infectious diseases. key: cord-286472-pqtem19t authors: mcfee, r.b. title: middle east respiratory syndrome (mers) coronavirus date: 2020-07-28 journal: dis mon doi: 10.1016/j.disamonth.2020.101053 sha: doc_id: 286472 cord_uid: pqtem19t nan ) (1) was isolated from a patient who died from severe pneumonia and multi-organ failure in saudi arabia (2, 3) . this newly identified respiratory viral illness was caused by a novel coronavirus, which was initially designated as human betacoronavirus (2) (3) (4) (5) , but was eventually named middle east respiratory syndrome coronavirus (mers cov). reminiscent of, and worth considering as a caution for greater vigilance towards emerging pathogens, the suddenness that sars cov emerged as a new cause of severe pulmonary illness, has been replicated in this new aggressive respiratory illness mers cov. unlike sars cov, it has not caused the thousands of cases over a short period of time. but it also differs from sars cov in that it carries a much higher case fatality rate, and causes more severe illness (6, 7) . unlike sars which seems to have gone quiescent, new mers cases have been reported well after the initial outbreak, including december 2019 (7b) . since september 2012 there have been cases reported in 27 countries across 4 continents, with most human cases occurring in saudi arabia (map 1 and 2) (6) . while the greatest spike in cases occurred near 2014, new cases to date continue to be reported. consider by the end of 12/19 the number of mers cases 2492, with 858 deaths, resulting in a case fatality rate of 34.43% (7b, 7c) . earlier data from world health organization (who) as of 12/05/16 there have been 1917 laboratory confirmed cases of mers cov since 09/12, reported from 27 countries (maps 1 and 2), resulting in 677 deaths, yielding a significant case fatality rate (~35%) (6); a fatality rate for a coronavirus that is significantly greater than that associated with sars cov delta (~9%), at least among confirmed cases (6) (7) (8) . these data demonstrate mers continues to cause illness, though thankfully not at pandemic rates in the same manner as covid-19. as with other cov, including sars cov and covid-19, the exact epidemiology, including the magnitude of mild or asymptomatic illness, remains unknown. that said, among those presenting with mers, males over 60 yrs of age seem to be at higher risk of severe disease symptoms and death among those infected. the risk associated with age seems consistent with covid-19 findings as well. abnormalities associated with mers cov include thrombocytopenia, lymphopenia, leukopenia, elevated serum lactate dehydrogenase (ldh), elevated aspartate aminotransferase (ast), elevated alanine aminotransferase (alt), along with abnormal renal function tests (5, 29, 30) . superinfection from viral or bacterial causes has been found in some patients and may complicate the course of the disease. severe cases of mers may require intensive care and mechanical ventilation, with a relatively large number of patients progressing to respiratory or renal failure. a retrospective radiology review of ct findings in laboratory confirmed cases of mers cov noted that cough, fever, and dyspnea were the most common presenting symptoms. three of the seven subjects died (31) . the high case fatality rate associated with mers cov has sparked interest in the role of virus on the immune system. one area of interest is the potential for cytokine storm, which is also found in covid-19 patients, and currently being studied extensively (31b) . some studies have shown mers cov is associated with an attenuated interferon (ifn) response, and does not induce inflammatory cytokines (7, 32, 33 chest xrays should be obtained early in the course of progressive pulmonary illness ( figure 2 ) (31). radiographic findings may include unilateral or bilateral patchy densities or opacities, interstitial infiltrates, consolidation, and pleural effusions. rapid progression to acute respiratory failure, acute respiratory distress syndrome (ards), refractory hypoxemia, and extrapulmonary complications (acute kidney injury requiring renal replacement therapy, hypotension requiring vasopressors, hepatic inflammation, septic shock) has been reported. key to appropriate testing is a thorough history and physical. it is important to consider multisystem function as well as pulmonary status in patients with severe respiratory illness, including suspected mers cov, especially those returning from regions where aggressive pathogens are noted. involving infectious disease and pulmonologist specialists early on, as well as good critical care management are essential. as noted earlier, laboratory findings at admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels (ldh). renal and hepatic function tests, at least for baseline information are worth obtaining. in the ajlan study (31) , all seven patients had lymphopenia. elevated creatinine developed after admission in all patients. ast also were elevated in all patients. of note, co-infection with other respiratory viruses and a few cases of co-infection with communityacquired bacteria at admission has been reported in mers cov patients. not surprisingly, nosocomial bacterial and fungal infections have also been reported in mechanically-ventilated patients. according to clinical experience reported to cdc, mers-cov virus can be detected with higher viral load and longer duration in the lower respiratory tract compared to the upper respiratory tract, and has been detected in feces, serum, and urine. however, very limited data are available on the duration of respiratory and extrapulmonary mers-cov shedding. as mentioned earlier, aggressive testing, isolation, and management are critical in treating highly pathogenic coronaviruses, especially mers, which among patients who present for medical care, has a high case fatality rate, extrapulmonary involvement in some cases, and rapid deterioration. this includes early radiographic studies (7b). according to the aljan radiology study (31) , chest xray findings were most notable for airspace and interstitial opacities with mers cov. radiographic findings such as airspace opacities are nonspecific, whereas the interstitial changes were described as reticular or reticulonodular. total lung opacification and thickening of the bronchovascular markings have been reported as well. previous chest ct findings with mers have been reported as bilateral patchy or extensive opacities (5, 7b) . not unexpected, imaging that had features consistent with acute respiratory distress syndrome were typically identified in sicker patients, and can be found in covid-19 patients as well (table 1 ). in aljan (31) mers cov the most common findings on ct were shows that airspace opacities on ct are common in patients hospitalized with mers-cov infection. airspace opacities are suggestive of an organizing pneumonia pattern. it was also described in h1n1 influenza a viral infections. in most of our patients, ground-glass opacities were more extensive than consolidation. septal thickening and pleural effusions also were demonstrated. importantly, tree-in-bud pattern, cavitation, and lymph node enlargement were not seen in our cohort. chest xrays and ct scanning ( figure 2 ) are recommended depending upon the presentation, and clinical course. in the aljan study (31) the most common ct finding in hospitalized patients with mers -cov infection reveals predominantly subpleural and basilar airspace changes, with more ground glass opacities than consolidation. it is important to note this is a small study. nevertheless this pattern is consistent with organizing pneumonia. patients recently returning from the middle east, presenting with significant respiratory illness, with ct findings of peribronchial region abnormalities, organizing pneumonia, should be considered for mers cov infection, and if possible, queried about international travel and occupational exposures. 27-year-old man with middle east respiratory syndrome patient was a smoker who was healthy otherwise. ct was performed 8 days after admission, and 20 days after onset of symptoms. patient was eventually discharged. lower lung ct image show large right lower lobe and small focal left lower lobe subpleural consolidations the cdc provides case definitions. the reader is requested to review periodically this site for updates as the mers cov situation can change (8, 34) . although emergency testing can be possible for suspected asymptomatic cases, the cdc guidelines for sending samples for laboratory confirmation are predicated upon a combination of clinical and epidemiological factors to identify persons under investigation (pui) table 1 . the other two categories per cdc are: a confirmed case is a person with laboratory confirmation of mers-cov infection. confirmatory laboratory testing requires a positive pcr on at least two specific genomic targets or a single positive target with sequencing on a second. laboratory confirmation of mers cov can be obtained by real time polymerase chain reaction (rt pcr) a probable case is a pui with absent or inconclusive laboratory results for mers-cov infection who is a close contact 3 of a laboratory-confirmed mers-cov case. examples of laboratory results that may be considered inconclusive include a positive test on a single pcr target, a positive test with an assay that has limited performance data available, or a negative test on an inadequate specimen. nb the reader is advised to check for updated testing procedures with the cdc (35) . this is an updated version of the interim guidance document issued by the centers for disease control and prevention (cdc) january 2014 based on input from public health partners, healthcare providers, professional organizations, and others. cdc will continue to update the document as necessary to incorporate new information that increases our understanding of mers-cov. updates: minor changes were made to clarify specimen type and collection procedures. of note -respiratory specimens should be collected as soon as possible after symptoms beginideally within 7 days. however, if more than a week has passed since symptom onset and the patient is still symptomatic, respiratory samples should still be collected, especially lower respiratory specimens since respiratory viruses can still be detected by rrt-pcr. for example -if symptom onset for a pui with respiratory symptoms was less than 14 days ago, a single serum specimen, an np/op specimen and lower respiratory specimen should be collected for cdc mers rrt-pcr testing. 1. if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was 14 or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. 2. if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was 14 or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. for short periods (≤ 72 hours), most specimens should be held at 2-8°c rather than frozen. for delays exceeding 72 hours, freeze specimens at -70°c as soon as possible after collection (with exceptions noted below). label each specimen container with the patient's id number, specimen type and the date the sample was collected. broncheoalveolar lavage, tracheal aspirate, pleural fluid collect 2-3 ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. have the patient rinse the mouth with water and then expectorate deep cough sputum directly into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. nasopharyngeal swab and oropharyngeal swab (np/op swab) use only synthetic fiber swabs with plastic shafts. do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate some viruses and inhibit pcr testing. place swabs immediately into sterile tubes containing 2-3 ml of viral transport media. np/op specimens can be combined, placing both swabs in the same vial. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. nasopharyngeal swab -insert a swab into the nostril parallel to the palate. leave the swab in place for a few seconds to absorb secretions. swab both nasopharyngeal areas. oropharyngeal swab (e.g., throat swab) -swab the posterior pharynx, avoiding the tongue. nasopharyngeal wash/aspirate or nasal aspirate collect 2-3 ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. for serum antibody testing: because we do not want to delay detection of mers infection and since the prevalence of mers in the us is low, serologic testing on a single serum sample collected 14 or more days after symptom onset may be beneficial. this is in contrast to serologic testing for many other respiratory pathogens which require collection and testing of acute and convalescent serum specimens. serologic testing is currently available at cdc upon request and approval. please be aware that the mers-cov serologic test is for research/surveillance purposes and not for diagnostic purposes -it is a tool developed in response to the mers-cov outbreak. contact cdc's emergency operations center (eoc) (770-488-7100) for consultation and approval if serologic testing is being considered . a single serum specimen collected optimally during the first 10-12 days after symptom onset is recommended. note: the kinetics of mers-cov are not well understood. once additional data become available, these recommendations will be updated as needed. the minimum amount of serum required for mers-cov testing (either serologic or rrt-pcr) is 200 µl. if both mers-cov serology and rrt-pcr tests are planned, the minimum amount of serum required is 400 µl (200 µl for each test). serum separator tubes should be stored upright for at least 30 minutes, and then centrifuged at 1000-1300 relative centrifugal force (rcf) for 10 minutes before removing the serum and placing it in a separate sterile tube for shipping (such as a cryovial). refrigerate the serum specimen at 2-8°c and ship on ice-pack; freezing and shipment of serum on dry ice is permissible. children and adults: collect 1 tube (5-10 ml) of whole blood in a serum separator tube. a minimum of 1 ml of whole blood is needed for testing pediatric patients. if possible, collect 1 ml in a serum separator tube. specimens from suspected mers cases must be packaged, shipped, and transported according to the current edition of the international air transport association (iata) dangerous goods regulations. shipments from outside of the united states may require an importation permit that can be obtained from cdc. specimens should be stored and shipped at the temperatures indicated above. if samples are unable to be shipped within 72 hours of collection, they should be stored at -70°c and shipped on dry ice. when shipping frozen specimen from long distances or from international locations, it is best to use a combination of dry ice and frozen gel ice-packs. the gel ice-packs will remain frozen for a day or two after the dry ice has dissipated. all specimens must be pre-packed to prevent breakage and spillage. specimen containers should be sealed with parafilm® and placed in ziplock bags. place enough absorbent material to absorb the entire contents of the secondary container (containing primary container) and separate the primary containers (containing specimen) to prevent breakage. send specimens with cold packs or other refrigerant blocks that are self-contained, not actual wet ice. this prevents leaking and the appearance of a spill. when large numbers of specimens are being shipped, they should be organized in a sequential manner in boxes with separate compartments for each specimen. saturday delivery is planned, special arrangements must be made with the shipping company. there are no commercially available or fda approved therapeutics specifically designated as mers cov antivirals. as of 2020 there remain a variety of candidate therapeutics in development against covid019 specifically, highly pathogenic coronaviruses such as sars and mers, and coronaviruses in general, including those that cause "common cold-like" symptoms, but none have emerged as fully capable of treating these viruses with the same consistency as neuraminidase antivirals have against influenza. and even in that context there are some clinical considerations, and limitations. there continues to be ongoing research into repurposing antivirals that are approved for other clinical indications such as hiv, influenza, and other illnesses, as possible treatments -monotherapy or in combination, against covid-19 and other coronaviruses. brief updates are provided below. covid-19 has reenergized research into greater study of the pathogenicity of coronaviruses, and newly or better described characteristics of highly pathogenic coronaviruses has led to increased insights into additional antiviral and vaccine targets. significant research into a wide array of therapeutic approaches is undergoing at unprecedented levels internationally. as will be seen in the covid-19 therapeutics section of this article, multiple options are being clinically tested that warrant consideration for the clinician faced with treating covid-19 patients. the mainstay of therapy for mers cov remains supportive care, especially respiratory care, while managing circulatory, renal, hepatic and neurological function, as well as protecting against secondary infections. although attempted in both the sars cov and early mers cov outbreaks, immune based interventions -interferon -resulting in equivocal outcomes. non human primate studies using ifn a2b and ribavirin against mers cov demonstrated improved outcomes but it is worth noting treatment was initiated soon after viral infection initiated; this is unlikely going to be the case with human infection, where a delay in presentation to health care, or delayed diagnosis will likely preclude the same rapidity of treatment that occurred in the study (6 -8, 32, 33) . the role for interferon in mers cov remains unclear; clinical trials are needed to better characterize successful strategies. but the limited number of cases make such studies difficult. in addition to various interferon treatment protocols, ribavirin -a potent nucleoside analog -has been utilized against rna viruses with varying degrees of success (7,32,33, 33b, 33c) . unfortunately ribavirin presents a risk for adverse effects including hemolytic anemia. interferon is not without side effect risk either. the early use of corticosteroids in sars cov infected patients resulted in increased viral load, critical care/intensive care unit admission, and death (7, 33c). other approaches to cov include neutralizing antibodies from convalescent plasma or hyperimmune globulin; these may hold some promise in reducing the cfr (33c, [36] [37] [38] . convalescent plasma was shown to decrease mortality in sars cov patients if provided to patients within 14 days of illness (36) . however in order for this to be effective, rapid diagnosis of patients, and survivors is required in order to obtain, and utilize these interventions. towards that end, a network for convalescent plasma is being assembled, allowing safety, effectiveness and logistic feasibility testing. (37) . that said, to date no host derived interventions have been shown to consistently confer significant benefit to severely ill mers cov patients via controlled study. while to date there are no antivirals currently licensed for use against mers cov, some candidate drugs are in development. two categories of candidates showed early promise. an adenine analogue that can be incorporated into viral rna, which can disrupt virus replication, and has shown in vitro activity against mers cov, as well as benefit against ebola in non human primates (nhps). also there is a nucleoside analogue for treatment of filoviruses, cov, and other rna viruses. researchers are working on other molecular approaches to treat cov (7, 39, 40) . ribavirin is a guanine, oral nucleoside analogue, which inhibits viral rna-dependent rna polymerase. in trials exploring the use of ribavirin against mers, it was often in combination with other therapeutics, including interferon. evidence revealed it did not confer significant clinical benefit on outcomes or viral clearance (33c, 41, 42) . it has shown some in vitro activity against sars, where high concentrations/high doses were required to inhibit viral replication (1.2 g to 2.4 g po every 8 hours), and combination therapy via intravenous or enteral administration (33b, 33c, 43) . studies on the use of ribavirin as a treatment for sars were either inconclusive in terms of clinical benefit, or suggested possible harm referable to adverse events, which included hepatic and hematologic deleterious effects (33c, 43). ribaviran as a potent nucleoside analogue has been, and continues to be used with varying degrees of success against rna viruses, but there is the potential for adverse side effects including hemolytic anemia, metabolic derangements. interferon can also elicit adverse effects, although they have demonstrated value against viral infection (44) (45) (46) (47) . another repurposing of antivirals involved lopinavir-ritonavir combination therapy trialed against mers. the medication is an oral protease inhibitor (33b, 33c). early success with treating sars in 2003, using lopinavir/ritonavir and ribavirin was suggested (33b, 48) . mortality and need for intensive respiratory support was noted (33b, 48) . in an animal study involving marmosets, lopinavir-ritonavir or interferon beta -1b reduced viral load and improved lung pathology (33b, 49) . combination antiviral therapy for patients hospitalized with severe influenza was noted in some cases to confer greater results, for those with high viral loads at presentation (33b, 50, 51) . it is worth noting that studies have shown both sars coronavirus and mers coronavirus their viral loads peak at ~7 -10 days after symptoms begin compared to covid-19 coronavirus where viral loads seem to peak at the time of clinical presentation (33b, 52,53) . this adds another level of clinical challenge in terms of managing this new, highly pathogenic coronavirus, and underscores the importance of time sensitivity in the administration of potentially lifesaving medications. another promising approach includes monoclonal antibodies (mabs). mabs have already demonstrated benefit against certain cancers and autoimmune disorder management (7, 38) . to date the only pathogen for which there is a licensed mab is respiratory syncytial virus (rsv). who recommends standard precautions with all patients, especially those displaying early signs of respiratory illness, as the diagnosis -whether common cold, or mers cov or rsv or pertusis or other pathogen related infection. also droplet precautions should be added to standard precautions when providing care to such patients, including those with acute respiratory illness. although eye protection is recommended for probable or confirmed cases of mers cov, and airborne precautions for aerosol generating procedures, this would seem prudent even in cases clearly not mers cov or sars cov. from an infection control perspective, among the known human coronaviruses they are capable of surviving on environmental surfaces for up to 3 hours (11) . the sars and mers experiences, as well as previous hcov, underscore the threat of transmissibility; coronaviruses may be transmitted from person-to-person by droplets, hand contamination, fomites, and small particle aerosols. avoiding camels, especially dromedary camels, farms, as well as not consuming raw milk, urine, or meat are also worth considering. camels infected with mers cov may not appear ill. if contact with animals, especially dromedary camels cannot be avoided, strict hand washing is recommended. personal protection, including face/eye if working with camels or other animals. patients who must travel to the region are encouraged to present to a health care facility especially if fever and respiratory symptoms develop. according to a who update, multiple cases from the united arab emirates, qatar, oman, kuwait, and bahrain have links to dromedary camels. these patients were thought to be infected via contact with infected dromedaries or close primary cases (uae). a good practice for anyone who works with potential contaminants -whether in the us or middle east farm -remove work clothes that may carry materials from farms and animal contact (poster 1) (53). to date few cases have occurred in europe or the us. however with the emergence of medical tourism -cosmetic and advanced surgeries -patients who reside in the middle east may bring with them diseases endemic to the region, including mers cov. the astute clinician will be aware of potential importation of illness. as of 2020 there are sporadic cases of mers-cov, which continues to cause illness since its emergence in 2012, and with a mortality rate estimated at greater than 30%. mers still remains a potential outbreak threat, and is a dangerous disease causing pathogen. as with sars cov and other viral threats of public health concern, there is a great need for both prophylactic measuresvaccines, and more targeted therapies. there is significant research effort underway internationally to develop an effective vaccine. as with the sars cov, there are a variety of approaches. one attempt which shows early animal efficacy is by a vaccine candidate consisting of chimeric virus-like particles (vlp) expressing the receptor binding domain (rbd) of mers-cov. the researchers have fused canine parvovirus (cpv) vp2 structural protein gene with the rbd of mers-cov; it self-assembles into chimeric, spherical vlp (svlp). this svlp retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to cpv virions. immunization with svlp induced rbd-specific humoral and cellular immune responses in mice. svlp-specific antisera from these animals were able to prevent pseudotyped mers-cov entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. of note, interferon (ifn) -ifn-γ, il-4 and il-2 secreting cells induced by the rbd were detected in the splenocytes of vaccinated mice by elispot. mice given svlp or an adjuvanted svlp vaccine elicited t-helper 1 (th1) and t-helper 2 (th2) cell-mediated immunity. although early stage, resarch svlp displaying the rbd of mers-cov may become, or lead to an effective vaccine against mers-cov. human research is needed (55) . of note, several of these approaches will be applied to developing vaccines against covid-19, which will be discussed in the next section (33c). -or -a member of a cluster of patients with severe acute respiratory illness (e.g., fever 1 and pneumonia requiring hospitalization) of unknown etiology in which mers-cov is being evaluated, in consultation with state and local health departments in the us. and close contact 3 with a confirmed mers case while the case was ill. fever may not be present in some patients, such as those who are very young, elderly, immunosuppressed, or taking certain medications. clinical judgement should be used to guide testing of patients in such situations countries considered in the arabian peninsula and neighboring include: bahrain; iraq; iran saudi arabia; syria; the united arab emirates meters), or within the room or care area, of a confirmed mers case for a prolonged period of time (such as caring for, living with, visiting, or sharing a healthcare waiting area or room with, a confirmed mers case) while not wearing recommended personal protective equipment or ppe (e.g., gowns, gloves, nioshcertified disposable n95 respirator, eye protection); or b) having direct contact with infectious secretions of a confirmed mers case (e.g., being coughed on) while not wearing recommended personal protective equipment. see cdc's interim infection prevention and control recommendations for hospitalized patients with mers for detailed information regarding healthcare personnel (hcp) please review cdc interim u.s. guidance for monitoring and movement of persons with potential middle east respiratory syndrome (mers-cov) exposure for more information: call 800-cdc-info (232-4636) or visit www.cdc.gov/travel poster 1 (54) mers references 1. image of middle east respiratory syndrome (mers) coronavirus as isolated by the national institute of allergy and infectious diseases isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus in a patient transferred to the united kingdom from the middle east middle east respiratory syndrome coronavirus (mers -cov) announcement of the cornoavirus study group epidemiological, demographic, and clnical characteristics of 47 cases of midle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study world health organization (who) mers update and global summary treatment strategies for middle east respiratory syndrome coronavirus radiology perspective of coronavirus disease 2019 (covid-19): lessons from severe acute respiratory 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combination of interferon beta-1b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open label randomized, phase 2 trial pharmacologic treatments for coronavirus disease 2019 (covid-19); a review centers for disease control (cdc) mers clinical laboratory testing recommendations the effectiveness of vonvalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility, safety, clinical and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol a conformation dependent neutralizing monoclonal antibody specifically targeting receptor binding domain in middle east respiratory syndrome coronavirus spike protein nucloeotide prodrug gs-5734 is a broad spectrum filovirus inhibitor that provides complete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx4430 in a non-human primate model of filovirus infecton clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and metaaanalysis ribavirin and interferon therapy for critically ill patients with mers: a multicenter observational study sars: systematic review of treatment effects modjarrad k treatment strategies for middle east respiratory syndrome coronavirus treatment with interferon-alpha2b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha 2a or ifn beta 1 in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment withlopinavir/ritonavir or interferon b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset antiviral combinations for severe influenza efficacy of clarithromycin-naproxen-oseltamivir combination in the treatment of patients hospitalized for influenza a (h3n2) infection: an open-label randomized, controlled, phase iib/iii trial temporal profiles of viral load inposterior oropharyngeal saliva sample and serum antibody responses during infection by sars cov-2: an observational cohort study medical treatment of viral pneumonmia including sars in immunocompetent adult novel chimeric virus-like particles vaccine displaying mers-cov antiviral res key: cord-256806-g42n51n9 authors: khudhair, ahmed; killerby, marie e.; al mulla, mariam; abou elkheir, kheir; ternanni, wassim; bandar, zyad; weber, stefan; khoury, mary; donnelly, george; al muhairi, salama; khalafalla, abdelmalik i.; trivedi, suvang; tamin, azaibi; thornburg, natalie j.; watson, john t.; gerber, susan i.; al hosani, farida; hall, aron j. title: risk factors for mers-cov seropositivity among animal market and slaughterhouse workers, abu dhabi, united arab emirates, 2014–2017 date: 2019-05-17 journal: emerg infect dis doi: 10.3201/eid2505.181728 sha: doc_id: 256806 cord_uid: g42n51n9 camel contact is a recognized risk factor for middle east respiratory syndrome coronavirus (mers-cov) infection. because specific camel exposures associated with mers-cov seropositivity are not fully understood, we investigated worker–camel interactions and mers-cov seroprevalence. we assessed worker seroprevalence in 2 slaughterhouses and 1 live-animal market in abu dhabi, united arab emirates, during 2014–2017 and administered an epidemiologic survey in 2016 and 2017. across 3 sampling rounds during 2014–2017, we sampled 100–235 workers, and 6%–19% were seropositive for mers-cov at each sampling round. one (1.4%) of 70 seronegative workers tested at multiple rounds seroconverted. on multivariable analyses, working as a camel salesman, handling live camels or their waste, and having diabetes were associated with seropositivity among all workers, whereas handling live camels and either administering medications or cleaning equipment was associated with seropositivity among market workers. characterization of high-risk exposures is critical for implementation of preventive measures. m iddle east respiratory syndrome (mers) coronavirus (mers-cov) was first identified as a cause of severe respiratory tract infections in saudi arabia in october 2012 (1) . the clinical spectrum of mers ranges from asymptomatic infection to acute respiratory distress syndrome and death (2) . as of april 3, 2019, a total of 2,374 laboratory-confirmed cases of infection have been reported by 27 countries to the world health organization (who); the reported case-fatality rate is 35% (2) . all reported cases have an epidemiologic link to the arabian peninsula, and imported cases have been reported in europe, asia, north america, and africa. the united arab emirates has reported the third-highest number of mers cases since 2012 (3) . mers-cov is a zoonotic virus, and dromedaries (camels) are recognized as a major virus reservoir for spillover to humans (4) . multiple studies have isolated mers-cov or mers-cov rna from camels across the arabian peninsula and africa (5) (6) (7) (8) (9) (10) (11) . serologic studies of camels in the middle east and africa have revealed mers-cov seroprevalence of >90%-97% (8, (11) (12) (13) . in natural infection, camels have been found to shed mers-cov in respiratory secretions and to a lesser extent in stool (14, 15) . evidence of virus rna has also been found in milk collected by traditional milking procedures, which involve calf suckling as a stimulus for milk letdown (15) . epidemiologic links between infected camels and human mers-cov infections have been shown, with identical or nearly identical mers-cov genomes found in human cases and in camels with which they had direct contact (16) (17) (18) . also, a case-control study identified exposure to camels as a risk factor for human mers-cov infection (19) . human seroprevalence studies also support the association between mers-cov infection and camel contact; in saudi arabia mers-cov seroprevalence was found to be 15 times greater in camel shepherds and 23 times greater in slaughterhouse workers compared with the general population (20) . further studies have also shown high seroprevalence in specific occupational groups with various camel exposures (e.g., seropositivity was detected in 6.8% of a cohort of 294 camel workers in qatar [21] and in 53% of a cohort of 30 camel workers in saudi arabia [22] ). although multiple lines of evidence suggest camel exposure is associated with human mers-cov infection, the exact mechanisms of transmission are not fully understood. information on specific risk factors relating to camel interactions are needed to further understand how the virus might be transmitted from camels to humans and to guide interventions to prevent zoonotic transmission, including changes to camel management practices. because mers-cov vaccines are currently in development and have reported success in phase i clinical trials (23), knowledge of groups at risk for mers-cov infection might also be useful when considering future vaccine use. our study aimed to identify risk factors for mers-cov seropositivity among live-animal market and slaughterhouse workers. the study sites consisted of an open-air animal market and 2 slaughterhouses (1 commercial and 1 public). all 3 facilities housed camels, goats, sheep, and cattle ( figure 1 ). typically during the study period, approximately 460 persons worked at the market, 101 at the public slaughterhouse, and 29 at the commercial slaughterhouse. the market investigated in this study was linked to a human mers case in 2015 (24) . prior investigation showed a large diversity of mers-covs circulating among camels at the market; 109 (29%) of 276 screened camels had detectable mers-cov rna in nasal swab specimens in the spring of 2015 (25) . we conducted 3 rounds of worker serum sampling. the first round was conducted during may 11-14, 2014 , and the second round during march 23-april 1 and may 7-13, 2015. during the first 2 rounds of sampling, all available workers at the market and public slaughterhouse were requested to provide a serum sample as part of a public health investigation. we conducted a third round of serum sampling during september 22-october 5, 2016, and march 20-23, 2017 . the third round of sampling included workers at the market, public slaughterhouse, and the newly opened commercial slaughterhouse. all available workers were requested to provide serum samples, although participation was voluntary. some, but not all, workers were repeatedly sampled, when feasible, during multiple rounds. we administered an epidemiologic survey to all workers only during the third round of serum sampling in 2016 and 2017. no surveys were administered in 2014 or 2015. the survey consisted of questions covering worker demographics; occupational history; contact with various animal species; travel history; medical history; consumption of raw camel milk, raw camel meat, and camel urine; specific tasks performed with camels; types of personal protective equipment (ppe) worn; and handwashing practices (appendix 1, https://wwwnc.cdc.gov/eid/article/25/5/18-1728-app1.pdf). separate lists of questions covering specific camel tasks performed were asked of market and slaughterhouse workers because of the different nature of camel tasks among occupational groups. interviews were conducted in arabic by staff from the abu dhabi department of health. human serum samples were tested for mers-cov antibodies at the us centers for disease control and prevention (cdc) by using indirect elisas for nucleocapsid (n) and spike (s) proteins, followed by a confirmatory microneutralization test, as previously described (26) . samples were initially tested by using both n and s elisas as screening assays with serum diluted to 1:400. all serum samples with optical densities above assay cutoff were diluted serially, 4-fold, from 1:100 to 1:6,400, and used for endpoint titer determinations. serum samples that were positive by n or s elisa with titers at 1:400, 1:1600, or 1:6,400, plus 10% of samples negative by n or s elisa at these titers, were tested by using microneutralization with live mers-cov performed in a biosafety level 3 laboratory, as previously described (26) . in addition, we conducted confirmatory microneutralization tests on seronegative samples from any persons who showed a change in seropositivity status over time to confirm changes in seropositivity status. samples were considered positive if positive on n and s elisa or if positive on microneutralization. specimens near the limits of detection but not consistently above or below these limits were considered indeterminate. for the epidemiologic analysis, persons with an indeterminate result were considered seronegative. we used epi info 7 (https://www.cdc.gov/epiinfo) for data entry and r version 3.3.1 (https://cran.r-project.org/ bin/windows/base/old/3.3.1) for data analysis. we performed comparisons between prevalence of work practices by setting (market vs. slaughterhouse) by using the pearson χ 2 square test. we used univariable logistic regression to estimate odds ratios, 95% cis, and p values (wald test) for all associations between potential risk factors and seropositivity. we assessed associations between demographics, occupational history, contact with various animal species, consumption of camel products, travel history, and medical history with seropositivity for all workers. we separately tested associations between specific interactions with camels, types of ppe worn, and handwashing practices with seropositivity for stratified subgroups of market and slaughterhouse workers because of the different nature of work setting and standard practices between these 2 populations. we then performed additional exploratory data description by occupation on the basis of results of univariable analyses. we developed 3 multivariable logistic models to identify associations between risk factors and seropositivity. first, we constructed a model of risk factors common to all workers and then constructed occupationally stratified models (i.e., separate models for market workers and slaughterhouse workers) to model specific interactions with camels, ppe use, and handwashing practices. we combined or eliminated highly correlated variables, which were determined by condition indices and variance decomposition proportions. we reduced categorical variables to binary options if small group size was observed. we performed initial variable selection by using least absolute shrinkage and selection operator (lasso) and then tested person-variable significance by using the likelihood ratio test with a cutoff of p<0.05 within an ordinary logistic regression model. we then included age and number of years worked at current setting as potential confounders in all 3 final models. we excluded persons with missing data at the lasso stage but included them for the final logistic regression model. for the stratified market worker and slaughterhouse models, we also included variables significant in the all workers model but not directly relating to camel interactions (e.g., reported underlying conditions) in the final occupationally stratified models. we did not include significant variables directly relating to camel exposures in the all workers model in the stratified models because more specific camel risk practices were assessed in the stratified models. for market and slaughterhouse models, we tested interactions between significant risk practices and select ppe use and handwashing practices for a protective effect. we sampled 100 workers in round 1 (2014), 151 workers in round 2 (2015), and 235 workers in round 3 (2016 and 2017); overall mers-cov seroprevalence was 6% for round 1, 19% for round 2, and 17% for round 3. twenty-one persons had specimens taken at rounds 1 and 2, twenty-three at rounds 2 and 3, thirteen at rounds 1 and 3, and twenty-two at all 3 rounds ( figure 2 ). of 70 persons who were seronegative at their first sample, only 1 (1.4%, 95% ci 0.1%-8.8%) seroconverted: a 30-year-old man who was a cleaner at the public slaughterhouse tested negative at round 1 and positive at round 2. of 8 persons who were seropositive at their first sample, 1 (13%) was later found to be seronegative: a 28-year old man who was an administrative supervisor at the market was resampled between rounds 1 and 3. this person did not report handling camels or their waste and did not perform any tasks directly relating to camels. one additional person who had a positive serologic result at their first and second samples and an indeterminate result at their third sample was not subsequently evaluated for change in seropositive status. because some study participants might have had different medical record numbers across the 3 sampling rounds, we could not determine all potential seroconversions or losses of seropositivity, although we also performed matching by name and age. we compiled serologic results for all participants who ever tested positive (appendix 2, https://wwwnc.cdc.gov/eid/ article/25/5/18-1728-app2.pdf). in total, 235 persons both completed the epidemiologic survey and were sampled during round 3. one additional person completed the epidemiologic survey but refused serum sampling and was not included in any analyses. all 235 workers were men, and their median age was 35 years (range 19-64 years). the median number of years worked at the current settings was 6 (range 0.2-15 years). we observed no significant effect of age (p = 0.26) or years worked (p = 0.18) on seropositivity on univariable analysis. worker occupations were categorized into animal handlers (n = 16), camel salesmen (n = 37), other animal salesmen (n = 41), animal or waste transporters (n = 27), butchers (n = 65), cleaners (n = 26), veterinarians (n = 9), and other (e.g., supervisor, cashier, and tourist guide) (n = 14). salesmen only worked in the market, and butchers only worked in the slaughterhouses. the remaining occupations were found in both settings, but each person could only work at a slaughterhouse or the market. none of the workers reported working at any other job outside of the market or slaughterhouses, and the only animals reported present at home were poultry and stray cats. overall, 64 (44%) of 145 market workers had daily contact with camels or their waste, compared with 47 (52%) of 90 slaughterhouse workers (p = 0.28). certain ppe use and handwashing were more frequently reported by slaughterhouse workers than market workers. among slaughterhouse workers, 99% reported wearing a dust mask (equivalent to a surgical mask), compared with 21% of market workers (p<0.01). only 37% of slaughterhouse workers reported taking their work clothes home, compared with 97% of market workers (p<0.01). eighty-one percent of slaughterhouse workers reported washing their hands before and after each animal-related task, compared with 21% of market workers (p<0.01). ninety-three percent of slaughterhouse workers reported washing their hands at the beginning and end of the day, compared with only 56% of market workers (p<0.01). rates of seropositivity were higher among market workers (29 [20%] of 145) than among slaughterhouse workers (11 [12%] of 90), although this difference was not statistically significant on univariable analysis (p = 0.17). by occupation, camel salesmen and animal or waste transporters had significantly higher odds of seropositivity than the reference group of other salesmen (table 1) . univariable analyses showed that several characteristics were associated with seropositivity among all workers (table 1) , including handling camels or their waste daily. not all seropositive workers reported handling camels or their waste; 7 workers initially claimed they never handled camels or their waste, although 3 of these later reported that they contacted either camel equipment, viscera, or waste within the slaughterhouse. for the subgroup of market workers, univariable analyses revealed multiple camel exposures to be associated with seropositivity and 2 handwashing practices that were inversely associated with seropositivity (table 2 ). for the subgroup of slaughterhouse workers, no individual risk factors were associated with seropositivity (table 3) . because camel salesmen had the highest odds of mers-cov seropositivity, we summarized their frequency of specific camel exposures separately (figure 3 ). direct observation of camel salesmen in the market showed that most of their time was spent in the camel pens, including while they ate and rested, and direct handling of the animals occurred frequently (data not shown). for the multivariable model evaluating risk factors associated with seropositivity in all workers, the following variables remained in the final logistic regression model: handling camels or their waste daily (adjusted odds ratio [aor] 4.2, 95% ci 1.7-11.8), working as a camel salesman (aor 4.0, 95% ci 1.6-10.1), and self-reported diabetes (aor 6.2, 95% ci 1.2-30.3). all 3 factors significantly increased odds of seropositivity. for market workers, multivariable analysis resulted in a final model in which the following variables were each independently associated with seropositivity: handling live camels (aor 12.2, 95% ci 3.2-62.9), administering medications to camels (aor 3.4, 95% ci 1.1-11.2), and self-reported diabetes (aor 20.9, 95% ci 1.6-341.3). cleaning equipment was also significantly associated with seropositivity (aor 3.3, 95% ci 1.1-10.3); substituted for administering medication to camels, this factor produced a model with a near-identical fit along with the other risk factors. given that administering medications to camels was highly correlated with cleaning equipment, the statistical significance of both factors was lost if both factors were included in the model because of collinearity (ρ = 0.65). none of the select ppe and handwashing practices evaluated as interactions with risk practices showed a significant protective effect. no individual risk factors were significantly associated with slaughterhouse workers by multivariable analysis. our study investigated risk factors for mers-cov seropositivity in animal market and slaughterhouse workers at a site previously associated with zoonotic transmission of mers-cov. given the large number of camels present, including many young camels, and the mixing of camels from multiple sources, this site probably facilitates mers-cov transmission among camels. our results demonstrated a relatively high mers-cov seroprevalence in workers at this site, ranging from 6% to 19% at each round across all occupations. because we did not record occupation and other risk factors during the first 2 sampling rounds, we were unable to further assess reasons for the different seropositivity rates between sampling rounds. we found particularly high seroprevalence in specific occupational groups, namely camel salesmen (49%) and animal or waste transporters (22%). previous studies of workers with occupational exposure to camels have reported either lower seropositivity rates (e.g., 6.8% of 294 workers with occupational camel contact seropositive in qatar [21] and 2.3% of 87 camel shepherds seropositive in saudi arabia [20] ) or comparable seropositivity (e.g., 53% of camel workers positive in saudi arabia [22] ). our rates of seropositivity might underestimate actual exposure to mers-cov. previous studies have demonstrated that examining mers-covspecific t cells from mers patients is more sensitive than examining serum antibodies alone (27) . to examine t-cell responses, peripheral blood mononuclear cells must be collected, which was beyond the scope of our study. on multivariable analysis, we found that contact with camels or their waste, working as a camel salesman, and self-reported diabetes were all independently associated with seropositivity in all workers. because of small stratum size, belonging to other occupational groups could not be meaningfully explored as risk factors. diabetes has previously been shown to be a commonly reported underlying condition in mers cases (28) , has been associated with risk for infection in a case-control study (19) , and has been associated with increased risk for death in mers patients (29) . we found an association between diabetes and mers-cov seropositivity in a cohort with occupational exposure to camels. although persons with diabetes might be at increased risk for mers-cov infection, the association between diabetes, mers-cov infection, and the resulting antibody response is still not fully understood. however, because persons with diabetes are considered at high risk for developing severe disease from mers-cov infection, who recommends these persons take precautions when visiting farms or markets where camels are present, including avoiding contact with camels (3). among market workers, handling live camels and either administering medications to camels or cleaning equipment were practices associated with significantly increased risk for mers-cov seropositivity. given that administering medications to camels was highly correlated with cleaning equipment, neither factor was statistically significant if both were included in the model. the biological importance of these associations might therefore be difficult to interpret, because either or both risk factors could be statistically associated with mers-cov seropositivity and have an undefined strength of association. practices potentially associated with camel calves, such as milking or assisting with camel birth, were not associated with mers-cov seropositivity despite a higher prevalence of viral rna in camels <1 year of age compared with other ages (30) and a previously reported association between milking camels frequently and seropositivity (31) . however, these practices were not commonly reported by market workers in our study, limiting the power to detect an association with seropositivity. no specific work practices were found to be associated with seropositivity among slaughterhouse workers. compared with market workers, slaughterhouse workers had less exposure to live camels and a higher self-reported prevalence of potentially protective practices such as ppe use and frequent handwashing. although our multivariable analysis did not show a significant association between ppe use (e.g., wearing a dust mask and gloves) or handwashing practices and seropositivity, the small sample size might have restricted the power to detect interactions between ppe and camel exposures. because camel-to-human transmission of mers-cov is not fully understood, who recommends broad preventive measures for slaughterhouse and market workers, including wearing facial protection when feasible, washing hands before and after each animal-related task, and washing soiled work clothes and shoes at the work place to avoid exposing family members to soiled work clothing (3) . where feasible, increased use of such measures could be encouraged, particularly in market workers, to decrease risk for infection. because only a single human mers case has been reported in connection with the study site, our reported rates of seroprevalence suggest unrecognized transmission (and potentially unrecognized illness) at this site. however, because the length of time mers-cov antibodies persist is unknown (32), the time and place these infections might have occurred is unknown; transmission potential also exists in the united arab emirates outside of markets and slaughterhouses. whether infections were symptomatic is also unknown. participants were asked whether they had seen a healthcare provider for respiratory illness in the previous 12 months, but such reported illness was not associated with seropositivity, and multiple pathogens other than mers-cov could be responsible for any reported respiratory illness. despite these limitations, mers-cov was detected in camels at the market during our study period (25) , and an interim seroconversion was noted in 1 worker, suggesting active zoonotic transmission. taken collectively, our findings suggest an underestimated prevalence of human mers-cov infection in settings where the virus is circulating among camels, probably resulting from camel-to-human transmission. our study had additional limitations, including the overall sample size and limited number of subjects within specific substrata. concentration of camel interactions within particular occupational groups limited our ability to differentiate risk among specific camel interactions, despite our use of multivariable analysis. furthermore, because most persons reported interactions either daily or never, determining whether increased risk was associated with increased frequency of individual tasks was not possible. also, some mers-cov infections might not result in detectable antibodies, particularly when the infections are asymptomatic or mild (32) . persistence of detectable mers-cov antibodies after infection is not well-defined, limiting the ability of serologic testing to define previous infection. finally, because of incomplete linkage of study participants by medical record numbers across the 3 sampling periods, not all potential seroconversions or losses of seropositivity could be determined. in summary, our study found significantly increased odds of mers-cov seropositivity in persons with exposure to camels, in particular among those who handle live camels. odds of seropositivity were also significantly higher for camel salesmen, suggesting that preventive measures such as ppe use could focus on specific occupational groups, in addition to individual work practices. determining groups at highest risk for zoonotic mers-cov infection could also inform future vaccine trials in geographic regions where mers-cov is known to circulate. middle east respiratory syndrome coronavirus (mers-cov) is a novel cov known to cause severe acute respiratory illness in humans; approximately 40% of confirmed cases have been fatal. human-tohuman transmission and multiple outbreaks of respiratory illness have been attributed to mers-cov, and severe respiratory illness caused by this virus continues to be identified. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) world health organization. who mers-cov global summary and assessment of risk middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction risk factors for mers coronavirus infection in dromedary camels in mers coronaviruses from camels in africa exhibit region-dependent genetic diversity middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels human infection with mers coronavirus after exposure to infected camels, saudi arabia high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study occupational exposure to dromedaries and risk for mers-cov infection high prevalence of mers-cov infection in camel workers in saudi arabia inovio pharmaceuticals i. inovio's mers vaccine generates high levels of antibodies and induces broad-based t cell responses in phase 1 study man in germany dies of complications stemming from mers virus diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study korean society of infectious diseases. clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea acute middle east respiratory syndrome coronavirus infection in livestock dromedaries risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study mers-cov antibody responses 1 year after symptom onset we thank yassir eltahir for critical input during the conceptualization and implementation of this study. this investigation was considered a public health response by the abu dhabi department of health and cdc. dr. khudair is a senior officer in the communicable disease control and management section of the abu dhabi department of health. his research interests include mers-cov and tuberculosis. dr. killerby is an epidemiologist in the division of viral diseases, national center for immunization and respiratory diseases, cdc. her research interests include respiratory viruses such as mers-cov, human coronaviruses, and adenoviruses. key: cord-299519-hfgmmuy6 authors: alenazi, thamer h.; arabi, yaseen m. title: severe middle east respiratory syndrome (mers) pneumonia date: 2019-10-26 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.11488-6 sha: doc_id: 299519 cord_uid: hfgmmuy6 middle east respiratory syndrome (mers) is a viral respiratory infection, which ranges from asymptomatic infection to severe pneumonia and multiorgan failure, caused by a novel coronavirus named middle east respiratory syndrome coronavirus (mers-cov). majority of cases have been reported from saudi arabia. mers cases occur as sporadic cases or as clusters or hospital outbreaks. dromedary camels are thought to be a host for mers-cov. direct contact with dromedary camels within 14 days prior to infection was identified as an independent risk factor for mers. diagnosis of mers is based on a positive real-time reverse transcriptase polymerase chain reaction (rrt-pcr), obtained from a respiratory specimen. the mainstay of management of mers-cov infection is supportive care. there is no specific antiviral therapy for mers-cov infection at present, although several modalities of treatment options have been examined or are under investigation. middle east respiratory syndrome (mers) is a viral respiratory infection, which ranges from asymptomatic infection to severe pneumonia, caused by a novel coronavirus named middle east respiratory syndrome coronavirus (mers-cov). coronaviruses are a family of viral pathogens that could cause animal and human disease. mers-cov is closely related to the severe acute respiratory syndrome coronavirus (sars-cov) but from a different lineage. the objective of this chapter is to describe the epidemiology, virology, clinical manifestations, management and prevention of mers. between september 2012, until the end of may 2019, the world health organization (who) has been notified of 2374 laboratoryconfirmed case of mers-cov infection from 27 countries with 823 associated deaths resulting in a case fatality rate of 35%. saudi arabia has been the major reporting country with a total number of 2008 cases and 749 deaths (a case fatality rate of 37.3%) ( table 1) (world health organization, 2019, mar 31) . the virus was first isolated in june 2012 from respiratory specimens of a saudi patient from jeddah, saudi arabia, who presented with severe pneumonia that progressed to acute respiratory distress syndrome (ards), renal failure, multi-organ failure and eventually lead to death (zaki et al., 2012) . in the same week, a patient with a recent history of travel to the united kingdom (uk) in august 2015, with 16 cases across three hospitals, and seven deaths (43.7% case fatality rate) (payne et al., 2018) . smaller outbreaks continued to occur in 2016-18 although the number of patients and the magnitude of the outbreaks were less compared to earlier years, presumably due to better infection control practices and earlier identification of cases. transmission of mers-cov in humans occurs through animal-to-human transmission or, human-to-human transmission in the community. additionally, nosocomial transmission of mers-cov occurs frequently. all transmission described up-to-date, occurred in residents in or travelers to the arabian peninsula, or are traced to contact with patients with a history of recent travel to the arabian peninsula. animals seem to play an important role in the transmission of the mers-cov. earlier studies have suggested that bats might be the potential reservoir of mers-cov. this hypothesis that was based on the close proximity of mers-cov-phylogenetically-to tylonycteris bat coronavirus hku4 (ty-batcov hku4) and pipistrellus bat coronavirus hku5 (pi-batcov hku5) (woo et al., 2012) . a study from saudi arabia, a phylogenetically mers-cov identical short gene segment, was detected in a fecal sample of one of the 29 captured bats near the home of a laboratory-confirmed mers-cov patient . however, live mers-cov has never been recovered from bats. further studies are needed to further establish the role of bats in transmission to humans including larger surveillance studies with full viral genome sequencing. epidemiologically, it seems unlikely that bats are the direct source of human cases, since none of the community-acquired laboratory-confirmed mers cases had clear bat exposure. dromedary camels are thought to be a host for mers-cov. direct contact with dromedary camels within 14 days prior to infection was identified as an independent risk factor for mers (gossner et al., 2016) . camel-human transmission was also suggested in a 44-year-old, previously healthy man from jeddah, saudi arabia who was admitted to the intensive care unit (icu) with severe mers pneumonia, and died 15 days after admission. the patient had owned a herd of 9 camels and used to visit them daily until 3 days prior to his admission. four out of the nine camels were sick with nasal discharge, 1 week prior to the patient's onset of symptoms. the patient had significant contact with camels' excretions. respiratory specimens from the patient and one of his camels showed identical mers-cov full genome sequencing. moreover, serum antibodies for mers-cov were positive in both the patient and the camel, with the camel seropositivity preceded the patient's seropositivity suggesting that direction of transmission was from the camel to the patient. a large cross-sectional study from saudi arabia identified mers-cov infected patients who had a history of camel contact. the investigators obtained nasal swabs and serum samples from 584 dromedary camels and found that 12.6% of the studied camels were mers-cov polymerase chain reaction (pcr) positive, and 70.9% of them were mers-cov antibodies positive. furthermore, 10 of the full genome sequences of the camel mers-cov were identical to their contacted patients (kasem et al., 2018) . this data suggests an important role for camels in the transmission of mers-cov. however, in a cohort of 1125 patients with laboratory-confirmed mers, camel contact was reported only in 235 patients (20.9%), denied by 276 patients (24.5%), and not reported in the other 614 patients (54.6%) (conzade et al., 2018) . hospital-based outbreaks and community-based clusters described above suggest strongly that human-human transmission does occur. the transmission was more commonly observed in healthcare-based outbreaks, compared to community clusters. the number of close contacts who got infected by patients with confirmed mers-cov appears to be low, although, it was evident that some patients were spreading the infection to a disproportionally large number of individuals (super spreaders) (hui, 2016) . this phenomenon was clearly described in more than one outbreak. the first outbreak which identified the super spreader phenomena was the korean outbreak, in which a single imported index case resulted in a total of 186 cases. it was thought that 83% of transmission in the korean outbreak was linked epidemiologically to five super spreaders (korea centers for disease and prevention, 2015) the same phenomenon was also described in a large outbreak in riyadh, saudi arabia, where 6 out of the 130 cases, contributed to 58.7% of the transmission (alenazi et al., 2017) . however, it remains unclear if an asymptomatic individual who carries mers-cov can transmit the virus to others. the first family cluster was reported from riyadh, saudi arabia, where three laboratory-confirmed cases and one probable case were diagnosed, and two out of the four patients died . in a study that investigated 26 index cases of mers and their 280 household contacts, the secondary transmission rate was found to be 4% ([95% ci, 2 to 7] . as described above, transmission was more commonly seen in hospital-based outbreaks compared to family community transmission, particularly in emergency department (ed). this was clearly illustrated in the korean outbreak, where a single imported case had led to a total of 186 cases, 185 of which were nosocomial transmission (kim et al., 2017) the main identified reasons for hospitals-based transmission were over-crowdedness of ed, late recognition of suspected mers cases and inadequate infection control measures and proper isolation of suspected cases (stone et al., 2016) . environmental surfaces in hospitals is a potential source of transmission. in one study, a viable mers-cov was detected in 15 out of 68 surface swabs collected from patient's rooms, restrooms and common corridors (kim et al., 2016) . mers-cov is the sixth coronavirus that affects humans. it lies within the lineage c of the genus betacoronavirus (cov) in the family coronaviridae under the order nidovirales. it has close phylogenetic proximity to two bat coronaviruses, tylonycteris bat cov hku4 (ty-batcov-hku4) and pipistrellus bat cov hku5 (pi-batcov-hku5). like the other coronaviruses, it is an enveloped single-stranded rna virus which replicates in the host-cell cytoplasm. the size of its rna genome is approximately 30 kb. it has structural proteins, called the e, m, and n proteins, and membrane protein called the spike (s) protein, which plays an important role in the virus attachment and entry into the host cells. due to the large increase in the number of diagnosed cases in april 2014, there was a concern that mers-cov could have undergone mutation that led to increased virulence or transmissibility of the virus; however, this assumption was proven unlikely (drosten et al., 2015) . the pathogenesis and histopathology of mers-cov is poorly understood and understudied. post-mortem autopsies were rarely performed on mers patients due to cultural reasons in the arabian peninsula. most of the knowledge we have about the histopathology of mers-cov comes from in vitro, ex vivo, animal experiments and limited post-mortem reports. in a 33-year-old male, who died of mers-cov infection, post-mortem analysis of histopathology finding of pulmonary and extrapulmonary tissue were examined under transmission electron microscopy which showed necrotizing pneumonia, pulmonary diffuse alveolar damage, acute kidney injury, portal and lobular hepatitis and myositis with muscle atrophic changes. the brain and heart were histologically unremarkable. ultra-structurally, viral particles were localized in the pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells and macrophages infiltrating the skeletal muscles (alsaad et al., 2018) . in the beginning of the outbreak, the who had proposed a case definition for mers-cov infection for epidemiological purposes, that was last updated on july 27, 2018. the united states (us) center of disease control and prevention (cdc) and the saudi ministry of health (moh), each had developed a case definition for suspected, confirmed and probable mers-cov infection ( table 2) . in one of the earlier outbreaks in saudi arabia, the median incubation period for mers-cov infection was 5.2 days (95% ci 1.9-14.7 days) (assiri et al., 2013b) . similarly, in the south korean outbreak, in 2015, the median incubation period was 6.3 days (95th percentile 12.1 days) (korea centers for disease and prevention, 2015) . therefore, mers should be suspected in patients presenting with respiratory infection, and residence in or travel to the arabian peninsula within the last 14 days prior to onset of symptoms. most of reported mers patients have been in the adult age group. only 31 pediatric cases were reported, most of which were detected on contact tracing screening (42% were asymptomatic), and among symptomatic cases, presence of comorbidities like congenital disease were commonly present (al-tawfiq et al., 2016) . the mean age in one of study was 56.3 years . in another study, that described the epidemiological, clinical characteristics and demographics of 47 mers-cov infected patients, 82.9% of laboratory-confirmed cases were more than 40 years of age with a median age of 56 years. the male: female ratio was 3.3:1. eighty nine percent of patients required icu admission, and the median time to death was 14 days (ranging from 5 to 36 days) (assiri et al., 2013a) . one study from saudi arabia, have compared critically ill mers-cov patients with critically ill non-mers-cov patients, and had found that mers-cov patients tend to be younger, more likely to require mechanical ventilation and had higher mortality . there were eight reported mers-cov infection during pregnancy, from jordan, united arab emirates and saudi arabia, three of them ended with maternal death . in the beginning of the epidemic, the typical presentation of reported mers was severe pneumonia, with acute respiratory distress syndrome (ards) with or without acute kidney injury, but as the surveillance and testing had increased, milder or even asymptomatic cases have been described. in a cohort of 47 patients, with mers-cov infection, the clinical presentation were fever (93%), cough (83%), shortness of breath (72%), myalgia (32%), diarrhea (26%), sore throat (21%), vomiting (21%), abdominal pain (17%) and hemoptysis (17%) (assiri et al., 2013a) . a study that compared 330 critically ill mers-cov infected patients with 220 critically ill patients with non-mers severe acute respiratory infection (sari) found that mers patients were younger than non-mers sari patients (median [q1, q3] 58 [44, 69] vs 70 [52, 78] and were more likely to be males (68.2% vs 58.1%) and to be healthcare workers (9.7% vs 0.0%). chronic comorbidities were prevalent (any comorbidity, 80.3% in mers sari, 91.4% in non-mers sari). after onset of symptoms, mers-cov patients presented to er with a median of 5 days and admitted to icu after 7 days, which was 2 days longer compared to non-mers-cov sari patients. mechanical ventilation was required for 85.2% of patients with mers-cov patients. at the time of icu admission, patients with mers-cov were more likely to be hypoxemic, compared with non-mers sari patients (ratio of arterial oxygen partial pressure to fractional inspired oxygen-pao2/fio2: 106.3 [66.2, 160] vs 176 [104, 252] ) . respiratory distress syndrome) and direct epidemiologic link with a laboratory-confirmed mers-cov case and testing for mers-cov is unavailable, negative on a single inadequate specimen or inconclusive. a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or acute respiratory distress syndrome) that cannot be explained fully by any other etiology and the person resides or traveled in the middle east, or in countries where mers-cov is known to be circulating in dromedary camels or where human infections have recently occurred and testing for mers-cov is inconclusive. an acute febrile respiratory illness of any severity and direct epidemiologic link (2) many cases of mers present with gastrointestinal manifestations with or without respiratory symptoms. among the critically ill patients, the most described extra-pulmonary manifestations were acute kidney injury and shock (arabi et al., 2014) . very few patients were reported to have neurological symptoms, in addition to the pneumonia (arabi et al., 2015) . primary infections were more likely to be severe, as opposed to secondary cases. secondary mers infection tends to cause a milder or asymptomatic disease, however severe disease has been described. secondary cases are more likely to be younger with no comorbidities. asymptomatic infections have bee also described in patients with dromedary camel's contacts who were identified during surveillance (al hammadi et al., 2015) . mortality rates were reported to be higher in older age group, male gender and patients with comorbidities (assiri et al., 2013a) . numerous cases of mers occurred among healthcare-workers; leading in some of them to severe illness resulting in admission to icu. in a study that examined 32 critically ill healthcare-workers with mers, 43.75% were nurses and 25% were physicians. 34.4% were having comorbidities, mainly chronic kidney disease (15.6%). fever at presentation, was found in 30/32 (93.8%), cough in 25/32 (78.1%), and gastrointestinal symptoms in11/32 (34.4%). eight out of the 32 (25%) healthcare workers died (shalhoub et al., 2018) . among all hospitalized patients with severe mers pneumonia, the most commonly observed laboratory abnormalities were lymphopenia (34%), thrombocytopenia (36%) and raised lactate dehydrogenase (ldh) (49%). other abnormalities like leukopenia (14%), lymphocytosis (11%), raised aspartate aminotransferase (ast) (15%), raised alanine aminotransferase (alt) (11%), and raised lactate dehydrogenase (49%) were also observed (assiri et al., 2013a) . in a cohort of 330 critically ill mers-cov patients, leukopenia was observed in 20.2%, thrombocytopenia in 58.7%, raised alt in 56.3%, and raised ast in 86.8% . most of the reported symptomatic cases with severe mers pneumonia had abnormal chest-x-ray. abnormalities ranged from mild to extensive changes. peripheral ground-glass opacities were the most frequently found abnormality on cxr, in 55 studied case (das et al., 2015) other findings include, unilateral or bilateral airspace opacities, increased broncho-vascular markings, patchy infiltrates, interstitial changes, nodular opacities, reticular opacities, reticulo-nodular shadowing, pleural effusions, and ards pattern. among inpatients who had chest computed tomography scan (ct scan), the most frequent findings were peripheral and bibasilar opacities bilaterally. in patient presenting with severe pneumonia, mers should be suspected based on the presence epidemiologic links (residence or travel from the arabian peninsula especially if there is history of contact with camels, contact with a person infected with mers or working or being treated in a hospital where mers patients are managed). such links should lead to application of appropriate infection control measures (see below) and to initiate diagnostic work up for mers. diagnosis of mers is based on a positive real-time reverse transcriptase polymerase chain reaction (rrt-pcr), obtained from a respiratory specimen. nasopharyngeal or oropharyngeal swab of the upper respiratory tract are often used in patients who are unable to produce lower respiratory samples. however, lower respiratory samples (sputum, endotracheal aspirate, or bronchoalveolar lavage) are preferred as they generally have better yield. in patients with suspected mers, it is recommended to send more than one specimen since a negative test does not exclude the diagnosis. in a cohort of critically ill patients with mers pneumonia, the diagnosis of mers was based on samples from the nasopharynx in 167 of 311 (54%) and from the lower respiratory tract (sputum, endotracheal aspirates, or broncho-alveolar lavage) in 144 of 311 (46%). the diagnosis was established from the first sample in 76% of patients, from the second sample in 16% of patients and from 3 to 5 repeat samples in 8% of the patients. initial negative samples collected before positive ones were predominantly from the upper respiratory tract (81.5%) . several serological assays have been used including enzyme-linked immune sorbent assay (elisa) and immunofluorescence assay (ifa), which are typically used for screening, and neutralization techniques which are used for confirmation. a three different, indirect elisa have been developed and validated based on mers-cov nucleocapsid protein (n), spike (s) ectodomain (amino acids 1-1297) and s1 subunit (amino acids 1-725) (hashem et al., 2019) . a single positive serological test, in the absence of positive pcr is considered a probable case, in the setting of suspected mers-cov. however, a four-fold increase in mers-cov antibody titer by neutralization tests is considered a confirmed case. viral pathogens were identified in 5% of critically ill patients with mers pneumonia which included other coronaviruses, respiratory syncytial virus, and influenza a virus. bacterial co-infections are described in 18% of critically ill patients with mers pneumonia, with acinetobacter species, pseudomonas species, klebsiella pneumoniae and staphylococcus aureus being the most frequent isolates . there is no specific antiviral therapy for mers-cov infection up to date, although several modalities of treatment options have been tried or are under investigation. the mainstay of management of mers-cov infection is supportive care. patients with suspected severe mers pneumonia-cov infection might have other respiratory pathogens as a cause of their symptoms. therefore, the who recommends starting appropriate empirical antimicrobial therapy as soon as possible, to cover community acquired or nosocomial associated pathogens, based on the presentation from the community or the hospital and based on local epidemiology and guidelines, until the microbiological diagnosis is confirmed. supportive therapy is the mainstay of management of severe mers pneumonia, which includes mechanical ventilation, vasopressor support, and renal replacement therapy if needed. oxygen rescue therapy like extracorporeal membrane oxygenation (ecmo) has been used in patients with refractory hypoxemia. in one case-control study of patients with mers, the rescue use of ecmo compared to a matched control with no-ecmo was associated with reduced in-hospital-mortality (65% compared 100%) (alshahrani et al., 2018) . another retrospective study, found that critically ill healthcare workers who died because of mers were more likely to have received ecmo than not, probably because the severity of pneumonia that led to use of rescue therapy, rather than use of ecmo itself (shalhoub et al., 2018) . corticosteroids have been used frequently in mers patients. a study that accounted for time-varying confounding demonstrated that corticosteroid use was not associated with difference on mortality although it was associated with prolongation of viral rna shedding (arabi et al., 2018b) . data on other human coronaviruses, and in vitro activity of specific therapies were used to identify potential new therapy for mers-cov. examples of those include: combination of ribavirin and interferon, lopinavir-ritonavir, mycophenolate mofetil, convalescent -plasma, and, monoclonal and polyclonal antibodies ( table 3) . the efficacy of ribavirin/interferon combination was suggested to be promising in vitro and animal experiments and cell culture. in a study where two cell viral cultures lines grew mers-cov, high concentrations of ribavirin or interferon alpha 2 b were needed to inhibit viral replication, when each of the drugs was used alone, however, comparable inhibition was observed when combing them at a lower concentration (falzarano et al., 2013a) . similar findings were observed in rhesus macaques model of mers-cov infection. among animals who received combination of ribavirin and interferon alpha 2 b 8 hours after inoculation did not develop respiratory symptoms and had no or very minimal chest x-ray findings of infiltrate compared to the control group. also, the treated group had a moderately lower viral genome copies and fewer severe lung histopathological changes (falzarano et al., 2013b) . data on humans are based on retrospective studies. in retrospective cohort of 20 patients with severe mers-cov pneumonia, ribavirin and interferon combination therapy started at median day three after diagnosis, showed improved 14-day survival, compared to 24 patients who received only supportive therapy, however 28-day survival was not different between the 2 groups (omrani et al., 2014) . other retrospective studies showed no difference in mortality between patients treated with ribavirin interferon combination, and patients who received supportive therapy shalhoub et al., 2015) . the largest cohort study which adjusted for time-varying confounders showed that ribavirin with interferons (alpha 1a and 1b and beta 1a) was not associated with difference in mortality or viral shedding. none of the patients received interferon beta 1b (arabi et al., 2019) . lopinavir-ritonavir efficacy was studied in-vitro in animals with severe mers-cov infection, in which it showed favorable outcome (chan et al., 2015) . there is an ongoing randomized placebo controlled trial evaluating oral lopinavir-ritonavir in combination with subcutaneous interferon beta-1b in hospitalized patients with mers (nct02845843) (arabi et al., 2018a) . the use of passive immune therapy with convalescent plasma was suggested as a potential therapeutic option. a study that examined the feasibility of convalescent plasma therapy for mers was limited by the small pool of donors with sufficient titers of mers-cov antibodies which may be related to the short-lasting immune response . several monoclonal antibody preparations have been developed. humanized bovine transchromosomal polyclonal antibodies against mers-cov have been developed and undergone testing in a phase i trial (beigel et al., 2018) . a phase ii trial in humans is being planned. mycophenolate mofetil efficacy against mers-cov was suggested in vitro studies. however, it was associated with harm in a marmoset model (chan et al., 2015) . remdesivir (gs-5734) which is the monophosphoramidate prodrug of the c-adenosine nucleoside analog gs-441524, has recently been reported to inhibit sars-cov, mers-cov and bat-cov, in vitro. it has also been found to be therapeutic and prophylactic in sars-cov infected mouse modules (agostini et al., 2018) . most of the reported hospital-based outbreaks were attributed to lack of adherence to proper infection control practice, delayed suspected cases identification, and to overcrowded emergency room and inappropriate triage. addressing issues related to infection control practice and proper triaging of patients with suspected mers-cov, had resulted in a decline in the number and the magnitude of hospital outbreaks . the who and us cdc have published guidance for mers prevention in healthcare institutes. as per the who recommendations, patients who have probable or confirmed mers should be under contact and droplet precautions with eye protection. the patient should be under airborne precaution, when performing an aerosol generating procedure (agp) like tracheal intubation or bronchoscopy. the us cdc, on the other hand, recommends contact and airborne precautions for all suspected or confirmed mers-cov patients. viral shedding from respiratory secretions has been found to be at least 3 weeks from onset of symptoms. therefore isolation precaution should not be discontinued until a negative pcr is obtained. patients with suspected or confirmed mers-cov, who does not require admission, can be isolated at home. it is recommended to avoid contact with camels, both direct or indirect contact like consuming raw camel's milk or meat. this is particularly for high risk individuals, such as patients with heart failure, chronic lung disease and immunosuppression. people who have to be in contact with camels should observe infection control precautions, including washing hand before and after contact, and use of appropriate personal protective equipment's (ppe) when dealing of a suspected or confirmed infected camels. it is important to note that the infected camels may not be symptomatic or might only have mild symptoms. the saudi authorities had made certain measures to reduce camel-human transmission, like banning camels in the holy areas, and moving the camels markets outside the cities. the who did not place any travel restriction to any country that have reported mers-cov cases. saudi arabia, where most of the laboratory-confirmed cases have been reported, annually hosts millions of muslims to perform hajj and omrah (pilgrimage), with no documented related cases of mers to date. there were 2 dutch patients who developed mers after returning from 2014 hajj, but the two cases were thought to be acquired from a camel market and raw milk consumption rather than human-human transmission during hajj. table 3 summary of treatment options for mers-cov. ribavirin/interferon combination ribavirin/interferon showed efficacy in and in vitro and in a rhesus macaques model. data in humans are based on retrospective studies. the largest cohort that accounted for time-varying confounders did not demonstrate efficacy. there may be differences in efficacy among different interferons, as interferon beta-1b has the lowest inhibitory concentrations in vitro falzarano et al. (2013a) and falzarano et al. (2013b) lopinavir-ritonavir lopinavir-ritonavir showed efficacy in in vitro and in a marmoset model. it is being tested in combination with interferon beta-1b in a randomized controlled trial (mers-cov infection treated with a combination of lopinavir /ritonavir and interferon beta-1b (miracle), nct02845843) chan et al. (2015) convalescent plasma the feasibility of the option is limited due to the paucity of donors arabi et al. (2015) monoclonal antibodies several monoclonal antibodies exist with promising efficacy in in vitro and in animal studies polycolonal antibodies demonstrated promising efficacy in in vitro and in animal studies. phase i trial has been completed. plans for phase ii trial are undergoing beigel et al. (2018) mycophenolate mofetil efficacy has been suggested in vitro but harm in a marmoset model chan et al. (2015) remdesivir this new drug has promising efficacy in in-vitro and in animal studies. phase i trial has been completed. phase ii trial is ongoing in ebola survivors agostini et al. (2018) there is no licensed human vaccine for mers-cov till now, however, many experimental candidate vaccines are under development. another approach is to vaccinate camels, as the source of infection for many human cases, and good progress has been made in this area (alharbi, 2017) . as of end of december 2018, the global case fatality rate for mers-cov infection was reported as 35.3% (806/2279). it is thought this number overestimates the case fatality rate of the disease, because milder and asymptomatic cases are likely to be underrepresented in the reported cases. this was suggested in a study that estimated the number of undetected human symptomatic cases to be 62% (cauchemez et al., 2014) . in a cohort of 47 mers-cov infected patients, case fatality rate was higher with increasing age (assiri et al., 2013a) . in another study that studied 939 mers-cov infected patients, independent risk factors for mortality were, age more 80 years, underlying cardiac comorbidity or cancer, and healthcare acquisition of the virus (alsahafi and cheng, 2016) . in a south korean cohort of 159 patients, risk factors for death were older age and underlying comorbidities (majumder et al., 2015) . coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department vaccines against middle east respiratory syndrome coronavirus for humans and camels histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-beta1b (miracle trial): study protocol for a randomized controlled trial corticosteroid therapy for critically ill patients with middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group transmission of mers-coronavirus in household contacts an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection development and validation of different indirect elisas for mers-cov serological testing super-spreading events of mers-cov infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia mers outbreak in korea: hospital-to-hospital transmission extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications middle east respiratory syndrome coronavirus outbreak in the republic of korea mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome coronavirus in bats, saudi arabia family cluster of middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah-a link to health care facilities ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study critically ill healthcare workers with the middle east respiratory syndrome (mers): a multicenter study unique case of papillary fibroelastoma originating from the right interatrial septum middle east respiratory syndrome (mers patient with new strain of coronavirus is treated in intensive care at london hospital genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 middle east respiratory syndrome coronavirus (mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia key: cord-297062-dmiplvt2 authors: almekhlafi, ghaleb a.; albarrak, mohammed m.; mandourah, yasser; hassan, sahar; alwan, abid; abudayah, abdullah; altayyar, sultan; mustafa, mohamed; aldaghestani, tareef; alghamedi, adnan; talag, ali; malik, muhammad k.; omrani, ali s.; sakr, yasser title: presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients date: 2016-05-07 journal: crit care doi: 10.1186/s13054-016-1303-8 sha: doc_id: 297062 cord_uid: dmiplvt2 background: middle east respiratory syndrome coronavirus infection is associated with high mortality rates but limited clinical data have been reported. we describe the clinical features and outcomes of patients admitted to an intensive care unit (icu) with middle east respiratory syndrome coronavirus (mers-cov) infection. methods: retrospective analysis of data from all adult (>18 years old) patients admitted to our 20-bed mixed icu with middle east respiratory syndrome coronavirus infection between october 1, 2012 and may 31, 2014. diagnosis was confirmed in all patients using real-time reverse transcription polymerase chain reaction on respiratory samples. results: during the observation period, 31 patients were admitted with mers-cov infection (mean age 59 ± 20 years, 22 [71 %] males). cough and tachypnea were reported in all patients; 22 (77.4 %) patients had bilateral pulmonary infiltrates. invasive mechanical ventilation was applied in 27 (87.1 %) and vasopressor therapy in 25 (80.6 %) patients during the intensive care unit stay. twenty-three (74.2 %) patients died in the icu. nonsurvivors were older, had greater apache ii and sofa scores on admission, and were more likely to have received invasive mechanical ventilation and vasopressor therapy. after adjustment for the severity of illness and the degree of organ dysfunction, the need for vasopressors was an independent risk factor for death in the icu (odds ratio = 18.33, 95 % confidence interval: 1.11–302.1, p = 0.04). conclusions: mers-cov infection requiring admission to the icu is associated with high morbidity and mortality. the need for vasopressor therapy is the main risk factor for death in these patients. electronic supplementary material: the online version of this article (doi:10.1186/s13054-016-1303-8) contains supplementary material, which is available to authorized users. middle east respiratory syndrome coronavirus (mers-cov) is a novel betacoronavirus that was first reported in september 2012 [1] . by january 6, 2016, a total of 1626 laboratory-confirmed cases of infection with mers-cov, including at least 586 related deaths, had been reported to the world health organization [2] . although mers-cov infections have been reported from 26 countries around the world, the majority of cases have originated in saudi arabia, south korea, the united arab emirates, jordan and qatar [3] . interhuman mers-cov transmission occurs in community and healthcare settings [4] [5] [6] [7] [8] . the exact source and mode of transmission of mers-cov to humans remains uncertain. however, mers-cov circulates among dromedary camels in africa and the middle east with occasions of documented camel-human inter-transmission [9] . there have been several reports outlining the clinical features and outcomes of patients with mers-cov infection [7, [10] [11] [12] [13] [14] [15] . however, very few have focused on critically ill patients in intensive care units (icu) [16] [17] [18] . there is therefore a need for more data to understand the various clinical and prognostic aspects of this potentially lethal disease, particularly for the most severe cases that require admission to the icu. we performed a retrospective study to describe the clinical features and outcomes of patients admitted to our icu with laboratory-confirmed mers-cov infection. the study was approved by the institutional review board of prince sultan military medical city (11159 riyadh, saudi arabia), a large tertiary-care referral center in riyadh, saudi arabia. informed consent was waived due to the retrospective, anonymous nature of data collection. we included all patients aged 18 years or more with confirmed mers-cov infection who were admitted to our 20-bed mixed medico-surgical icu between october 1, 2012 and may 31, 2014. all icu patients with a confirmed diagnosis of mers-cov were registered in a special logbook. for the purpose of the current study, all patients' records were reviewed by a senior intensivist (s. hussain, a. alwan, a. abudayah, s. altayyar, m. mustafa, t. aldaghestani, a. alghamedi, a. talag or m. malik). clinical data and laboratory parameters from confirmed cases of mers-cov were transcribed onto specially developed case record forms. these included the initial manifestations of respiratory infection, the clinical picture on admission to the icu, laboratory indices of organ failure, radiographic findings, interventions during the icu stay, treatment modalities, and final outcome. the acute physiology and chronic health evaluation ii (apache ii) score was calculated from the data obtained within 24 hours of admission to the icu [19] . the sequential organ failure assessment (sofa), score, calculated daily by the physician in charge of the patient, was also noted [20] . since the first reported cases of mers-cov in saudi arabia in september 2012, all suspected cases in our institution are strictly isolated and nasopharyngeal swabs are obtained for initial screening. deep respiratory samples (tracheal aspirates or bronchoalveolar lavage fluid) are obtained from all patients admitted to the icu with suspected respiratory infections, in addition to blood samples to perform cultures and polymerase chain reaction for common respiratory viruses and atypical microorganisms. urinary samples were obtained to detect legionella antigens in only two patients (legionella infections are not common in saudi patients). cultures of tracheal aspirates are analyzed quantitatively and bacterial counts of at least 10 5 colony-forming units are considered positive. these investigations are repeated in the icu whenever secondary infections are suspected. clinical specimens aimed at detecting possible mers-cov infection are processed and analyzed at the national reference laboratory of the saudi ministry of health. mers-cov infections are identified using real-time, reverse transcription polymerase chain reaction (rt-pcr). the standard assays target amplifications of the upstream e protein (upe gene) and open reading frame (orf)1a; both need to be positive to confirm infection, otherwise another sample is required to confirm the diagnosis [21] . the sample requires 2 days of processing for the final results to be available. routine laboratory testing in our icu includes complete blood counts, coagulation profile, electrolytes, renal function, liver profile and arterial blood gases. these parameters are measured on admission to the icu and at least once daily thereafter (at 6:00 am) throughout the icu stay. all patients with suspected or confirmed mers-cov infection were isolated in single rooms, either on the hospital floor or in the icu. patients were admitted to the icu according to the guidelines of the society of critical care medicine for icu admission, discharge, and triage [22] . patients were classified into four categories according to their icu admission priority: priority one comprised critically ill patients who were unstable and need intensive treatment and monitoring, with significant likelihood of recovery; priority two were stable patients who required intensive monitoring because of the possibility of decompensation; priority three were unstable patients who had a low likelihood of recovery because of the severity of acute disease or because of comorbidities; priority four were those who had little or no anticipated benefit from icu admission. patients classified as priority one and two and most of those classified as priority three were admitted to our icu or full critical care services were mobilized and provided for in the isolation ward until a bed was available in the icu. priority four patients were not admitted to the icu and remained in the isolation ward. general ward patients with mers-cov infection were transferred to the icu if their condition deteriorated or organ failure developed. the infection control precautions recommended by the saudi ministry of health guidelines were strictly implemented to prevent possible transmission of mers-cov to other patients or to the healthcare staff [23] . supportive treatment was provided according to our standard operating procedures and in accordance with the surviving sepsis campaign guidelines [24, 25] . antiviral therapies, such as oseltamivir, and ribavirin/interferon alfa-2a, were prescribed at the discretion of the attending physician. protective lung ventilation was applied in mechanically ventilated patients. prone positioning was considered in some patients with severe refractory hypoxemia. extracorporeal membrane oxygenation (ecmo) and high-frequency oscillation were also available as a last resort, when considered necessary by the attending physician. statistical analyses were performed using spss statistics 19 for windows (ibm corp., armonk, ny, usa). the kolmogorov-smirnov test was used to verify whether there were significant deviations from the normality assumption of continuous variables. nonparametric tests of comparison were used for variables evaluated as not normally distributed. difference testing between groups was performed using student's t test, mann-whitney test, chi-square test and fisher's exact test, as appropriate. friedman's test was used to assess the time course of organ function. to identify the risk factors for death in the icu, we performed multivariable logistic regression analyses. due to the relatively small number of deaths in our study, we adjusted only for the severity of illness on admission to the icu (apache ii score) and the degree of organ dysfunction as assessed by admission sofa score. potential risk factors for icu mortality were selected among the demographic characteristics, comorbidities, mode of acquisition of mers-cov, initial manifestations, procedures and therapies, and superimposing infections. variables yielding p <0.2 in the univariate analysis, apa-che ii score and sofa score were included in a multivariable logistic regression analysis. these variables were introduced separately into multivariable models including apache ii and sofa scores on admission to the icu. adjusted odds ratios (or) and 95 % confidence of interval (ci) were computed. none of the covariates simultaneously introduced in a multivariable model were collinear. data are presented as mean ± standard deviation (sd), median value (25th-75th interquartile range [iqr]) or number (%), as appropriate. all statistics were two-tailed and a p < 0.05 was considered statistically significant. during the observation period, 70 cases with confirmed mers-cov infections were diagnosed in our institution [11] (fig. 1) ; 21 patients were managed in the hospital ward, 18 patients were admitted to other icus or received critical care service in the ward, and 31 patients were admitted to our icu (12 between october 1, 2012 and december 31, 2013 and 19 between january 1 and may 31, 2014). patients were admitted to our icu because of respiratory failure (pao2/fio2 < 250 mmhg). the mean age of the patients admitted to our icu (n = 31) was 59 (sd 20) years and 22 (71 %) were males. the characteristics of these patients on admission to the icu are shown in table 1 . eighteen (58.1 %) patients had community-acquired mers-cov infection, while for 13 (47.9 %), including two healthcare staff, infection was acquired in the hospital. twenty-seven patients (87.1 %) had at least one comorbidity. the median number of concomitant comorbid conditions was three (iqr: 2-4). initial clinical manifestations had occurred at a median of 2 days (iqr: 2-4) prior to hospital admission. patients had been treated for a median of 5 days (iqr: 2-9) in general hospital wards before their admission to the icu. only four patients (12.9 %) were admitted to the icu on arrival at the hospital. cough and tachypnea were reported in all patients. other common initial symptoms were fever (87.1 %), abdominal pain (29 %), sore throat (25.8 %), and fatigue (25.8 %) (additional file 1). crackles (93.5 %), tachycardia (67.7 %), and rhonchi (32.3 %) were the most commonly identified initial physical signs. bilateral pulmonary infiltrates were present in the chest x-rays of 24 (77.4 %) patients and lobar infiltrates in six (19.4 %). only one patient had a normal chest x-ray at the time of admission to the icu. on admission to the icu, no patients had microbiologically proven co-existing bacterial pneumonia. secondary infections, as evident from positive quantitative cultures of deep tracheal aspirates, occurred in 18 (58.1) patients within a median of 3 days (iqr: 3-8) after admission to the icu. the most commonly isolated microorganisms were acinetobacter baumannii (25.8 %), only four (12.9 %) patients had positive blood cultures; acinetobacter baumannii (n = 2), escherichia coli (n = 1), methicillin-resistant staphylococcus aureus (n = 1), and vancomycin-resistant enterococcus species (n = 1). invasive mechanical ventilation was applied in 27 (87.1 %) patients during the icu stay; 18 (58.1 %) within 24 hours of admission to the icu, and 14 (45.5 %) patients received noninvasive ventilation (table 2) . eleven (35.5 %) patients were treated with high-frequency oscillation and five (16.1 %) with prone positioning. only one patient received ecmo. the ventilatory parameters are presented in additional file 3. vasopressor therapy using norepinephrine was initiated in 25 (80.6 %) patients (table 2) . oseltamivir was administered to 20 (64.5 %) patients for a median of 5 days (iqr: 3-5). combined ribavirin plus interferon alfa-2a therapy was used in 13 (41.9 %) patients ( table 2 ). all patients received at least one antimicrobial agent during the icu stay (additional file 2). antifungal therapy was only used in four of the five patients with positive cultures for candida but the necessity of this therapy is uncertain. the overall icu mortality rate was 74.2 % (n = 23). the median icu and hospital lengths of stay were 9 (iqr: 4-16) and 12 (iqr: 4-16) days, respectively. the major causes of death were hypoxemic respiratory failure (52.2 %) and refractory septic shock (26.1 %). one patient died from sudden cardiac arrest after icu discharge but while still in the hospital. furthermore, one patient died within 1 year after discharge from the icu because of septic shock related to an infected wound. only one patient was lost to follow-up after hospital discharge. the sofa score and glasgow coma scale (gcs) increased markedly over the first 2 weeks in the icu in the whole cohort, while other parameters of organ function remained largely unchanged (additional file 3). compared with those who were discharged alive from the icu, nonsurvivors were older, had higher apache ii and sofa scores on admission to the icu, and were more likely to require invasive mechanical ventilation and vasopressor therapy and to have been ventilated using highfrequency oscillation (tables 1 and 2 , and additional files 1 and 2). nonsurvivors had a persistently low pao2/fio 2 throughout the first 2 weeks in the icu, whereas survivors showed a slight increase over time (fig. 2) . after adjustment for the severity of illness and the degree of organ dysfunction, the need for vasopressors was the only independent risk factor for death in the icu (or 18.33, 95 % confidence interval 1.11-302.1, p 0.04) (additional file 4). the 31 critically ill patients with confirmed mers-cov infection in our cohort frequently had organ failure with an overall mortality rate greater than 74 %. comorbidities were common in this cohort of patients. not surprisingly, mortality in the icu was associated with older age, severe disease and organ failure. the need for vasopressor therapy was an independent risk factor of death in the icu. since the first reported case of mers-cov infection in 2012, several authors have described various cohorts of patients with this serious infection [8, 10-12, 14, 15, 26] . [16, 18] . our facility is a large tertiarycare medical center in riyadh, central saudi arabia. we herein provide a detailed account of the largest single cohort of critically ill mers-cov infected patients reported thus far. in agreement with previous reports from saudi arabia, comorbid conditions were common in our patients with mers-cov infections with a median of three comorbidities per patient [10, 11, 15, 17] . in contrast, only 54.8 % of the 186 individuals involved in the recent mers-cov outbreak in south korea had any preexisting chronic medical conditions [8] . however, only 29.6 % of patients in the korean outbreak were aged 65 years or older and nearly half (46.2 %) were caregivers or healthcare personnel [8] . the differences in the demographic characteristics of our cohorts and the mode of acquisition of mers-cov infection may explain, at least in part, the discrepancy in the patterns of associated comorbidities between the saudi and korean cohorts. the respiratory manifestations of mers-cov infection in our cohort were similar to those observed in previous reports from saudi patients [10, 11, 14, 15, 17] . cough and tachypnea occurred in all patients and 77 % of cases had bilateral pulmonary infiltrates, denoting severe respiratory illness, which required a median of 5 days to reach the peak of clinical deterioration such that icu admission and organ support therapy were required. gastrointestinal manifestations, such as abdominal pain, diarrhea, vomiting, and abdominal tenderness, were relatively common in our cohort. this was also a common finding in the previous literature in patients with mers-cov infection as well as those with severe acute respiratory syndrome (sars) [10, 11, 15, 17, 28, 29] . our data confirm previous studies that reported a high prevalence of nonrespiratory organ failure in critically ill patients with mers-cov [16, 17] . the mechanisms of organ dysfunction and failure in these patients are yet to be determined. cytokine dysregulation has been suggested to be involved in the pathophysiology of mers-cov-related organ failure. direct viral invasion may also occur as the virus was recovered from urine and stool in one patient [30] . in agreement with the results of the previous reports on critically ill patients with mers-cov infection [16] [17] [18] , more than 80 % of our patients received vasopressor support, underscoring the high prevalence of cardiovascular dysfunction in these patients, and suggesting that disturbances in tissue perfusion may also have been involved in the pathophysiology of the organ failure. lower rates of vasopressor support have been reported in patients with sars [10, 11, 15, 17, 28, 29] with, as a result, lower mortality rates than those reported in patients with mers-cov infections. even though overall mortality rate was high in our cohort, it is still comparable with rates reported in previous studies (58.3-64.3 %) [16] [17] [18] . in all studies, almost all patients had significant comorbidities and median apache ii scores of 25 or higher. we observed significantly higher apache ii and sofa scores in icu nonsurvivors compared to those who survived severe mers-cov infection, underscoring the strong association between mortality and the severity of disease. epidemiological analyses have suggested that mers-cov is unlikely to trigger sustained human epidemics at present [31, 32] . nevertheless, nosocomial outbreaks have resulted in considerable morbidity and mortality, in addition to disruption of medical services and substantial economic losses [9, 33, 34] . the most severe infections usually require icu admission, necessitate major resource utilization and result in high fatality rates. identifying possible risk factors for poor prognosis in patients with mers-cov infection is therefore crucial to enable appropriate allocation of healthcare resources and early transfer of high-risk patients to the appropriate medical facilities. our data show that the need for vasopressor therapy was an independent risk factor for death in the icu. indeed, the major causes of death in our study were hypoxemic respiratory failure and refractory septic shock, which confirm the role of respiratory and cardiovascular system failures as determinants of outcome in this population. this was also evident from the persistent hypoxemia observed in the nonsurvivors. to date, published data on the risk factors for poor prognosis specific to critically ill patients with mers-cov infection are lacking. in cohort studies of patients with any degree of severity of mers-cov infection, older age, diabetes, chronic renal failure, chronic respiratory disease, high viral load in lower respiratory tract samples, shorter incubation period and mers-cov viremia have all identified as independent predictors of mortality [11, 14, [35] [36] [37] . secondary respiratory infections occurred commonly in this cohort, predominantly with gram-negative bacteria. although candida species were frequently isolated, these are probably not relevant as respiratory pathogens and the necessity of antifungal therapy is uncertain. interestingly, acinetobacter baumannii, which is an emerging fatal infection in icu patients worldwide, was isolated from deep tracheal aspirates in one in four patients. this may explain, at least in part, the relatively high mortality rates in this cohort. specific therapeutic options for mer-cov infections are limited and their efficacy is not well established [38] . all patients in this report received antiviral treatment with either oseltamivir or combined ribavirin/interferon alfa-2a therapy; two patients received both. although a previous study from the same institution showed that combined ribavirin/interferon alfa-2a therapy was associated with significant improvement in survival at 14 days, this benefit was not maintained at 28 days after the onset of the disease [39] . the retrospective and observational nature of this study does not allow precise assessment of the efficacy of these therapies. in the absence of a vaccine or a specific treatment, prevention of viral transmission through adequate infection control methods is the mainstay in the management of mers-cov outbreaks. appropriate isolation of patients with suspected or proven infections is crucial. in view of the high fatality rates of these patients in the icu, it may be reasonable to closely monitor patients with suspected infections in the general wards for early signs of organ dysfunction to prevent unnecessary delay in the provision of intensive care services and reduce mortality rates in these patients. our study has some limitations. we included patients with confirmed mers-cov infection from one icu of a large medical center. possible variations in the geographic distribution of the disease and in local practice may hinder extrapolation of these data to other cohorts in saudi arabia and other countries. the relatively low number of patients may 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of critical care medicine infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012 multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome in the shadow of ebola middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart spread of mers to south korea and china mers coronavirus: diagnostics, epidemiology and transmission mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death, saudi arabia association between severity of mers-cov infection and incubation period therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) infection: how close are we? curr treat options infect dis ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study we would like to thank dr. hassane nijimi (free university of brussels) for the statistical revision of this study and dr. karen pickett for editorial assistance with the manuscript. the study was supported only by institutional funds. mers-cov infections requiring admission to the icu are associated with high morbidity and mortality rates. the need for vasopressor therapy is the main risk factor for death in these patients. this report describes the clinical features and outcomes of 31critically ill patients with confirmed middle east respiratory syndrome coronavirus (mers-cov) infection. patients with mers-cov infections frequently had organ failure, and mortality rates were greater than 72 %. the need for vasopressor therapy was an independent risk factor for death in the icu. the authors declare that they have no competing interests.authors' contributions gaa, ym, aso, mma, and ys conceived the study. sh, aal, aab, sa, mm, ta, aalg, at, and mkm participated in data collection. ys processed the data and performed the statistical analyses. ys and gaa drafted the manuscript. all authors read, revised, and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-329514-dnh3rx0q authors: kurono, yuichi; lim, david j.; mogi, goro title: middle ear and eustachian tube date: 2007-05-09 journal: mucosal immunology doi: 10.1016/b978-012491543-5/50092-9 sha: doc_id: 329514 cord_uid: dnh3rx0q nan despite the development of a variety of antibiotics and the improvement of sanitary conditions, otitis media (om), such as acute otitis media (aom) and otitis media with effusion (ome), is still one of the most prevalent infectious diseases in children and accounts for a significant medical cost. further, the bacteria that commonly cause om are increasingly showing antibiotic resistance. therefore, the development of a vaccine against those bacteria is considered an important goal for public health. many studies have demonstrated that immunologic reactions are involved in the occurrence of om. elevated specific serum antibodies, mainly igg, inactivate pathogenic bacteria and resolve the infection. infants with recurrent episodes of om have been shown to have significantly decreased igg antibody levels against pneumococcal capsular polysaccharide (prellner et al., 1989) . it has also been reported that children with recurrent om have decreased serum igg2 subclass levels (freijd et al., 1985) . these findings indicate that systemic humoral immunity protects the middle ear from bacterial infection and that enhancement of the immunity might be effective to prevent om. however, the protective efficacy of parenteral administration of pneumococcal polysaccharide vaccine was disappointing (karma et al., 1985) . the poor effect of the vaccine suggests that reformulation of the immunizing preparation and use of different routes of immunization, such as oral and intranasal immunization, may enhance the protective efficacy against om. mucosal immunization induces the production of secretory iga (s-iga) in external secretions via mucosal immune system. in earlier studies, mogi et al. (1974) isolated s-iga from pooled middle ear effusions (mees) and revealed that the antigenicity and subunit structure of s-iga are identical to those of s-iga obtained from other external secretions such as saliva, nasal secretion, colostrum, and bronchial fluid. ogra et al. (1974) demonstrated that specific antibody activity in mees against mumps, measles, rubella, and poliovirus is limited to s-iga. further, intraduodenal or intratracheal immunization induced antigen-specific igaforming cells in the tympanic mucosa (watanabe et al., 1988) . these findings suggest that the middle ear might be a potential organ to act as an effector site of the mucosal immune system. the purpose of this chapter is to review recent available data regarding the mucosal immune system equipped in the middle ear and microbiologic as well as immunologic aspects of om, and to discuss the efficacy of mucosal vaccines for om. by pcr and demonstrated that the incidence was significantly higher in patients with ome than that in controls. they also reported that p6 gene dna was detected in all nasopharyngeal secretions of patients with ome who had p6 gene dna in mees. those findings suggest that microorganisms in the nasopharynx, as well as those in the middle ear, play an important role in the pathogenesis of om. further, rayner et al. (1998) demonstrated the presence of bacterial mrna in mees by an rt-pcr-based assay. because bacterial mrna has a half-life measured in seconds to minutes, detection of bacteria-specific mrna would be evidence that metabolically active organisms are present. the results showed that all specimens having dna of h. influenzae detected by pcr but negative by conventional culture method were positive by rt-pcr, indicating the presence of viable, metabolically active, intact bacteria in some culture-negative cases of ome. recent studies regarding microbiology of om have focused on identification of bacterial adherence factors such as h. influenzae fimbriae (hif), high-molecular-weight (hmw) adhesion proteins, and pneumococcal surface adhesion a (psaa). hif is classified into the major (hifa) and minor (hifd and hife) subunits, and both the major and minor subunits were required for adherence of h. influenzae to oropharyngeal epithelial cells (van ham et al., 1995) . however, immunologic and structural characteristics vary among nthi (mccrea et al., 1998) . in contrast, hmw adhesion proteins, hmw1 and hmw2, are members of a family of highly immunogenic proteins and are common to 70% to 75% of nthi strains (barenkamp and st geme, 1996) , suggesting the possibility of developing vaccine against diseases caused by nthi including aom and ome. lipooligosaccharide (los) is known to be a virulence factor of nthi. demaria et al. (1997) examined the relative virulence of a parent nthi strain and two different los-deficient mutants by evaluating the ability of those strains to colonize the nasopharynx and to induce om subsequent to transbullar inoculation in chinchilla model. they found that the abilities of mutants to induce om and to persist in the middle ear were significantly decreased compared with the parent nthi. thus, the presence of intact los molecules appears to be critical to the virulence of nthi in inducing om. moreover, sun et al. (2000) reported that detoxified los-protein conjugates from nthi elicited a significant rise of anti-los antibodies with bactericidal activity and inhibitory activity of the adherence of nthi. those data indicate the availability of detoxified los as a vaccine for om. most cases of aom are preceded by a viral upper respiratory infection. okamoto et al. (1993) detected genomic sequences of rsv in the samples of mee obtained from patients with ome by rt-pcr. in those patients from whose nasopharynx rsv was isolated, the viral sequences were highly detectable in mees. recently, pitkaranta et al. (1998) reported that viral rna of human rhinoviruses, rsv, and coronaviruses was detected in 48% of mees, 62% of nasopharyngeal aspirate samples, and in 57% of bacteria-negative mee samples collected from children with aom at the time of diagnosis. heikkinen et al. (1999) also examined the prevalence of various respiratory viruses in mees of 456 children with aom. they detected rsv in 74% of samples, parainfluenza viruses in 52%, and influenza viruses in 42% of mee, suggesting that rsv was the principal virus invading the middle ear during aom. these findings highlight the importance of common respiratory viruses in predisposing young children to aom and in causing this disease. wadowsky et al. (1995) examined the relationship between viral infection and isolation of bacterial pathogens from the middle ear. they intranasally inoculated influenza viruses into adult subjects and found that s. pneumoniae was isolated from 15% of subjects on day 6, whereas the bacteria was not isolated before the virus challenge. belshe et al. (1998) reported that live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine reduced the incidence of aom during the epidemic of influenza. in an experimental study using mice, influenza a virus infection changed the glycoconjugate sequence of the surface of nasopharyngeal mucosa, and increased the colonization of h. influenzae and s. pneumoniae inoculated after the virus infection (hirano et al., 1999) . suzuki and bakaletz (1994) demonstrated the synergistic effects between adenovirus and nthi in inducing experimental om. the attachment of p5-fimbriated nthi to respiratory epithelial cells was significantly enhanced by rsv infection (jiang et al., 1999) . those results indicate that viral infection might be the sole etiology of om in some children and that preceding viral infection alters the defenses preventing bacterial colonization and invasion into the middle ear. serum antibodies, mainly igg, induced during om are pathogen specific. all patients with ome who lacked serum igg activity against the organism causing the second episode possessed the antibody activity against the first strain at the time of the second episode (faden et al., 1989) . on the other hand, yamanaka and faden (1993) reported that certain children, who are classified as otitis prone, do not respond well to p6, whereas non-otitis-prone individuals are more likely to develop a significant antibody response to p6. p6 is highly conserved among strains of nthi and serves as a target for bactericidal antibody. demaria et al. (1996) demonstrated that subcutaneous immunization of chinchillas with p6 elevated serum bactericidal antibody activity against homologous as well as a heterologous nthi isolate and reduced the incidence of nthi culture-positive middle ear fluids after post-transbullar challenge with live nthi. further, yamanaka and faden (1993) reported that the concentration of p6-specific igg in mee was directly related to the concentration of anti-p6 antibody in serum and that the concentration of p6-specific igg in mee was inversely related to the number of bacteria in mee. those findings suggest that systemic immune responses may account for the resolution of om. although specific igg antibodies are protective against the invasion of bacterial antigen into the middle ear, some evidence suggests that systemic immune system may be involved in inducing or sustaining mees. ueyama et al. (1997) reported that when chinchillas were inoculated with live s. pneumoniae after systemic immunization with killed bacteria of the same strain, 7 out of 20 animals developed chronic ome. these findings suggest that the formation of immune complexes induces immune-mediated om and is associated with the persistence of inflammatory reactions in the middle ear. the presence of antigen-specific iga antibodies in mees of patients with aom as well as ome suggests that the mucosal immune system in the middle ear plays a role in the pathogenesis of those diseases. liu et al. (1975) found that iga levels in mees were significantly higher in culture-negative samples than in culture-positive samples, and that bacterial recovery rate was related inversely to the increase in the levels of iga and igg in mees. sloyer et al. (1974) reported that specific igg, igm, and iga antibodies against causative pneumococcal serotype were found in 27% of mees of patients with aom, and that specific iga antibodies were detected more often in mees than in serum. on the other hand, yamanaka and faden (1993) measured antibody activities in mees to p6 of nthi and reported that igg specific to p6 was detected more frequently than igm, iga, and s-iga antibodies. these findings suggest that the local as well as systemic immunity is associated with the immune responses in the middle ear cavity and the pathogenesis of om. inflammatory cytokines such as il-1and tnf-α were found in mees of children with ome, and the levels of those cytokines were higher in younger children than in older children (yellon et al., 1991) . schousboe et al. (2001) investigated the relationship among microorganisms, endotoxin, and inflammatory mediators in ome and found positive correlation between endotoxin and inflammatory cytokines such as tnf-α and il-1β in culture-positive mees as well as in culture-negative ones, suggesting endotoxin-induced local production of tnf-α and il-1β in the middle ear. watanabe et al. (2001) further investigated the relationship between endotoxin and the production of il-1β. they developed a murine model of ome by intratympanic injections with endotoxin derived from nthi and with recombinant il-1β. anti-il-1 receptor antibodies inhibited the pathologic changes induced by endotoxin and by recombinant il-1β. these results suggest that il-1β is associated with the middle ear inflammation induced by endotoxin. further, il-1β induces the production of il-8, which is associated with the migration of neutrophils and the secretion of mucin from goblet cells (smirnova et al., 2002) . inflammatory cytokines were detected in nasopharyngeal secretions. healthy children, 1 to 3 years of age, were found to have significantly higher levels of il-1β and tnf-α in nasopharyngeal secretions compared with healthy adults. in contrast, children with recurrent episodes of om had significantly lower levels of il-1β than healthy children (lindberg et al., 1994) . the results indicate that nasopharyngeal cytokine activity is protective for middle ear infection and that the defect in cytokine production might be responsible for defective immune reactivity. the increase of iga antibodies in mees indicates the induction of mucosal immune response in the middle ear during om. mucosal immune response is regulated by immunoregulatory cytokines such as il-2, il-4, il-5, and tgf-β. il-2 and il-4 function primarily as general immune activators in immunoglobulin production and stimulate b cells already committed to igm, igg, or iga to differentiate into plasma cells. il-5 preferentially stimulates the proliferation and production of iga-b cells already committed to this isotype. tgf-β stimulates class-switching of igm-b cells to an iga phenotype. bikhazi and ryan (1995) investigated the expression of those immunoregulatory cytokines associated with the production of different antibody isotypes in experimental acute and chronic ome using in situ mrna hybridization. they found that cells producing il-2 and il-4, but not il-5, were present during acute ome. however, in chronic ome, il-2-and il-4-producing cells were less prevalent, but cells producing il-5 were numerous. cooter et al. (1998) demonstrated the presence of tgf-β in the middle ear, and showed that tgf-β1 and tgf-β2 levels in mees are elevated in association with a history of previous tympanostomy tube placements and chronic mucoid effusion. those results are consistent with the enhancement of igg production in acute ome and increased local production of iga during chronic ome. further, the findings indicate the induction and enhancement of mucosal immune responses during the inflammatory process in the middle ear. there are few immunocytes in the normal tubal and middle ear mucosa of humans and animals (ichimiya et al., 1990) and immune cell recruitment requires antigen stimulation (matsune et al., 1996) . however, the middle ear cavity of normal human and laboratory animals is sterile, although numerous immunocompetent cells as well as a variety of pathogens are found in the middle ear during om. these findings imply that there are highly effective antimicrobial defense systems, so called the innate immune system, protecting the tubotympanum other than the acquired immune system. the system consists of the mucociliary function of the tubotympanum and the molecules secreted by its epithelial cells, which create a highly effective barrier against invading pathogens and help to maintain the sterility of the middle ear cavity. included in the molecules of the innate immune system are lysozyme, lactoferrin, defensins, and members of the collectin family of the surfactant proteins such as sp-a and sp-d (bevins, 1999) . increased production of those molecules in mees suggests the importance in the pathogenesis of om. lysozyme has potent antibacterial properties because of its ability to disrupt bacterial cell walls, and it is an important participant in host defense at mucosal surfaces, pleural fluid, and in leukocytes (leitch and willcox, 1998) . although there are no data available as to the role of lysozyme in the protection of the tubotympanum, it is possible that tubotympanal lysozyme, together with other antibacterial factors, may protect these tissues. lim et al. (2000) demonstrated that lysozyme itself did not kill nthi, but preexposure of bacteria to lysozyme enhanced bactericidal activity of β-defensins. lactoferrin is an iron-binding glycoprotein found in the milk and exocrine secretions of mammals including those of eustachian tube and middle ear mucosa. lactoferrin synergistically interacts with immunoglobulins, complement, and neutrophil cationic proteins against gram-negative bacteria by inflicting damage to the outer membrane (ellison, 1994) . lactoferrin is also active against nthi, and the proteolytic activity was first discovered when strains of nthi were cultured in the presence of human milk whey (qiu et al., 1998) . recently, it has been demonstrated that the proteolytic activity of lactoferrin causes hap adhesin of nthi strains to lose their ability to adhere to epithelial cells and removes most of the iga protease inhibitor from the outer membrane of those bacteria (plaut et al., 2000) . defensins are endogenous antimicrobial peptides widely distributed among the plant and animal kingdoms. the peptides represent elements of the ancestral immune system predating lymphocytes and immunoglobulins (lehrer and ganz, 1999) . the vertebrate defensin family can be divided into αand β-defensins. β-defensins are primarily expressed by the epithelial cells of skin, kidneys, and tracheobronchial lining of nearly all vertebrates, where they can be released upon microbial invasion or upregulated by stimulation with lipopolysaccharide and tnf-α (stolzenberg et al., 1997) . boe et al. (1999) demonstrated the presence of human β-defensin-1 mrna in the tympanic membrane and meatal skin by rt-pcr and in situ hybridization studies. recently, moon et al. (2002) showed the expression of β-defensin-2 in the human middle ear mucosa and human middle ear epithelial cell line. further, they demonstrated that its expression is induced by proinflammatory stimuli such as il-1α, tnf-α, and lipopolysaccharide. although the involvement of these defensins in the defense of the eustachian tube and middle ear is yet to be proven, the potent microbicidal properties of defensins induced in the human middle ear epithelial cell line suggests that they may play a role in the pathogenesis of om. sp-a and sp-d can bind to bacteria, fungi, and viruses via their carbohydrate groups causing them to agglutinate. this promotes phagocytosis by alveolar macrophages (nepomuceno et al., 1997) . several investigators have demonstrated the expression of surfactant proteins sp-a and sp-d in the eustachian tube and middle ear epithelium (dutton et al., 1999; paananen et al., 1999) . although the role of sp-a and sp-d in the defense of the tubotympanum remains to be proven, it is likely that a deficiency in these molecules may contribute to the pathogenesis of om. mucins are also associated with the innate immune system of tubotympanum. mucins are high-molecular-weight glycoproteins that constitute the major component of mucus secretions in all mucosal surfaces including the eustachian tube and middle ear. mucus secretions protect and lubricate the epithelial surface and trap bacteria and viruses for mucociliary clearance. the mucus blanket and the periciliary fluid seromucin are critical for the proper functioning of the mucociliary system. moreover, mucins act as receptors for a variety of bacteria including h. influenzae and are associated with bacterial adherence to epithelial surface. thus, mucins play an important role in the mechanical clearance of bacteria. because the pathogens colonizing in the nasopharynx invade into the middle ear mucosa via the eustachian tube and cause om, the nasopharynx is equipped with a specific immunologic defense system. several studies have demonstrated that increased levels of s-iga in nasopharyngeal secretions are associated with the decrease of the colonization of middle ear pathogens such as s. pneumoniae and h. influenzae in the nasopharynx (kurono et al., 1991; harabuchi et al., 1994) . the inhibitory effect of iga antibodies against nasopharyngeal colonization was confirmed by animal experiments kurono et al., 1999) . oral as well as intranasal immunization of mice with outer membrane proteins of nthi significantly enhanced antigen-specific iga antibody titers in nasal wash and the ability to clear the same strain of live nthi inoculated into the nasopharynx. on the other hand, subcutaneous immunization affected neither iga antibody titer in nasal wash nor nasopharyngeal colonization by nthi. demaria et al. (1996) also reported that parenteral immunization with p6 did not alter the extent or duration of nasopharyngeal colonization by nthi. these results indicate that iga antibodies in nasopharyngeal secretions inhibit the adherence of middle ear pathogens to nasopharyngeal mucosa and facilitate the clearance of those bacteria from the nasopharynx. it is well acknowledged that s-iga antibody is induced by the mucosal immune system, which is anatomically and functionally divided into two compartments: inductive sites and effector sites. the inductive sites include the gut-associated lymphoid tissue (galt), bronchus-associated lymphoid tissue (balt), and nasal-associated lymphoid tissue (nalt). iga precursor b cells in the inductive sites are primed and activated by mucosal immunization and disseminate throughout the mucosal effector sites such as nasal mucosa, nasopharyngeal mucosa, and salivary glands via the common mucosal immune system (mcghee and kiyono, 1994) . nalt has been identified as an inductive site of rodents, and the embryologic as well as immunologic characteristics recently have been investigated (fukuyama et al., 2002) . in humans, the adenoids and palatine tonsils are considered to act as nalt. quiding-järbrink et al. (1995) showed that intratonsillar vaccination induced a substantial immune response such as igg and iga antibody-producing cells in tonsils. further, primary immunization in one palatine tonsil followed by a second immunization of both palatine tonsils evoked a larger immune response in the primed tonsil. sakamoto et al. (1998) demonstrated that the numbers of p6-specific iga-producing cells in the adenoids were significantly correlated with iga antibody titers in nasopharyngeal secretions. those findings suggest that human palatine tonsils and adenoids serve as not only inductive sites but also effector sites of the mucosal immune system in the nasopharynx. recently, several studies demonstrated that middle ear mucosa has a function as an effector site of the mucosal immune system. kodama et al. (2000) and suenaga et al. (2001) examined the characteristics of the lymphocytes in the middle ear mucosa of mice at the single-cell level after intranasal immunization with p6 together with cholera toxin (fig. 88.1) .they found the induction of p6-specific iga-producing cells into the middle ear mucosa and a similar cell population of t/b cells, cd4 + /cd8 + cells, and αβ t cells between the middle ear mucosa and nasal mucosa after intranasal immunization. further, the presence of a certain amount of γδ t cells were observed in both middle ear and nasal mucosa, whereas no γδ t cells were found in the other lymphoid tissues such as nalt, cervical lymph nodes, spleen, or peyer's patches. those findings suggest that the middle ear mucosa has the same function to nasal mucosa as an effector site of the mucosal immune responses induced by intranasal immunization. kodama et al. (2000) further investigated the t-cell responses in the middle ear mucosa of mice after intranasal immunization with p6. the data showed an increase of memory t cells as well as p6-specific iga-b-cell in the middle ear mucosa after intranasal immunization. in vitro stimulation with p6 resulted in a proliferation of purified cd4 + t cells obtained from the middle ear mucosa of immunized mice, and those t cells expressed th2 cytokine mrna. moreover, cd4 + t cells enabled iga-b cells derived from nalt to differentiate into iga plasma cells in the middle ear mucosa. those findings suggest that antigen-specific iga-b cells primed in nalt might home to the middle ear mucosa and differentiate into iga-producing plasma cells with the help of th2 cells recruited to the middle ear mucosa. prevention of om is complicated by the multifactorial nature of the disease. predisposing factors include eustachian tube dysfunction, early nasopharyngeal colonization by nthi, immune dysfunction, and others. further, the bacterial strains causing om, such as s. pneumoniae and nthi, are extremely diverse. therefore, effective vaccines for om must possess surface epitopes of microbial antigens that are common among strains and able to elicit protective antibodies. because nthi lack capsular polysaccharide, antigenic determinants of nthi are outer membrane proteins. among the outer membrane proteins, p6 is a highly conserved peptidoglycan-associated lipoprotein present in all strains of nthi. kyd et al. (1995) have demonstrated that intra-peyer's patch immunization with p6 followed by an intratracheal boost induced p6-specific cd4 + t-helper cell proliferation in lymphocytes isolated from the mesenteric lymph nodes and increased antibody response in both serum and bronchoalveolar lavage fluid. however, the absence of enhanced pulmonary clearance in some animals suggests that the route of immunization might have affected the induction of mucosal immune responses in respiratory tract responsible for the pulmonary clearance of nthi. to develop a useful vaccine delivery system to prevent om, kurono et al. (1999) compared the mucosal immune responses in mice immunized with nthi antigen intranasally, orally, and intraperitoneally. the results showed that antigen-specific iga antibody titers in nasal washes and the numbers of antigen-specific iga-producing cells in nasal passages were significantly increased in intranasally immunized mice when compared with the other groups (fig. 88.2) . cytokine assay showed that ifn-γ, il-2, and il-6 were predominantly produced in intranasal immunization, suggesting that th2-as well as th1-type cells were involved in the induction of antigen-specific immune response. furthermore, bacterial clearance of a homologous strain of nthi from the nasal tract was significantly enhanced in the nasal immunization group. these findings suggest that intranasal immunization is an effective vaccination regimen for the induction of antigen-specific mucosal immune responses in the upper respiratory tract. hotomi et al. (1998) investigated the immune responses induced by intranasal immunization with p6.they found that intranasal immunization of mice with p6 together with cholera toxin evoked a good mucosal iga as well as a systemic igg response against p6 and increased the number of anti-p6-specific antibody-producing cells in the nasal mucosa of immunized mice. the protective effects of intranasal immunization were confirmed in vitro by the enhancement of nasopharyngeal clearance of nthi and the inhibition of adherence of nthi to cultured human epithelial cells. in another experiment, they reported that intranasal immunization with recombinant p6 together with cholera toxin also oral cavity, upper airway, and head and neck induction of h. influenzae (hi)-specific antibody-forming cells in mucosal tissues after intranasal, oral, intratracheal, and intraperitoneal (ip) immunization with outer membrane complex of hi. antigen-specific iga antibodies were predominantly induced in nasal passage by intranasal immunization. modified from kurono et al. (1999) . induced nthi-specific mucosal and systemic immune responses (hotomi et al., 2002) . sabirov et al. (2001) assessed the effect of intranasal immunization for the protection against nthi-induced om in mice. they found that the clearance of nthi from the middle ear was remarkably enhanced and the incidence of nthi-induced experimental om was significantly reduced by intranasal immunization with p6 together with cholera toxin (table 88 .1). further, immunized mice showed lower concentrations of tnf-α in mees. these findings strongly suggest that p6 is a good potential vaccine candidate and intranasal vaccination with p6 affords protection against om caused by nthi. recently, the efficacy of a newly developed live attenuated intranasal influenza vaccine was investigated in 1602 children (belshe et al., 1998) . the vaccine reduced not only culture-positive influenza virus but also the incidence of febrile aom, indicating that prevention of viral infection is an effective way to prevent the development of aom. further, the results suggest that intranasal vaccine might be a useful vaccine 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in the middle ear were supported in part by grant-in-aid for general scientific research (b) (2) 14370548 from the ministry of education, science and culture of japan. key: cord-323133-gdg50omp authors: buzatto, g. p.; tamashiro, e.; proenca-modena, j. l.; saturno, t. h.; prates, m. c.; gagliardi, t. b.; carenzi, l. r.; massuda, e. t.; hyppolito, m. a.; valera, f. c. p.; arruda, e.; anselmo-lima, w. t. title: the pathogens profile in children with otitis media with effusion and adenoid hypertrophy date: 2017-02-23 journal: plos one doi: 10.1371/journal.pone.0171049 sha: doc_id: 323133 cord_uid: gdg50omp objectives: to evaluate the presence of viruses and bacteria in middle ear and adenoids of patients with and without otitis media with effusion (ome). methods: adenoid samples and middle ear washes (mew) were obtained from children with ome associated with adenoid hypertrophy undergoing adenoidectomy and tympanostomy, and compared to those obtained from patients undergoing cochlear implant surgery, as a control group. specific dna or rna of 9 respiratory viruses (rhinovirus, influenza virus, picornavirus, syncytial respiratory virus, metapneumovirus, coronavirus, enterovirus, adenovirus and bocavirus) and 5 bacteria (s. pneumoniae, h. influenzae, m. catarrhalis, p. aeruginosa and s. aureus) were extracted and quantified by real-time pcr. results: 37 ome and 14 cochlear implant children were included in the study. at the adenoid, virus and bacteria were similarly detected in both ome and control patients. at the middle ear washes, however, a higher prevalence of bacteria was observed in patients with ome (p = 0.01). s. pneumoniae (p = 0.01) and m. catarrhalis (p = 0.022) were the bacteria responsible for this difference. although total virus detection was not statistically different from controls at the middle ear washes (p = 0.065), adenovirus was detected in higher proportions in adenoid samples of ome patients than controls (p = 0.019). conclusions: despite both ome and control patients presented similar rates of viruses and bacteria at the adenoid, children with ome presented higher prevalence of s. pneumonia, m. catarrhalis in middle ear and adenovirus in adenoids when compared to controls. these findings could suggest that these pathogens could contribute to the fluid persistence in the middle ear. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 otitis media with effusion (ome) is a common childhood disease characterized by the presence of fluid in the middle ear, with no symptoms and/or signs of acute inflammation [1, 2] . in the united states, approximately 90% of all children develop an episode of ome before they reach school age, mainly between the ages of 4 months and 6 years [2] . the presence of ome is associated with severe negative impact on child development, including hearing loss with long-term consequences for speech and language acquisition, poor school performance, and imbalance issues [1] [2] [3] . moreover, children with ome are five times more susceptible to develop acute otitis media than controls [4, 5] . the pathogenesis of ome is still not fully understood. epidemiological data suggest that the pathogenesis of ome is multifactorial, involving anatomical, immunological, genetic, microbial, and environmental factors [6] [7] [8] [9] . however, it is widely accepted that the dysfunction of the eustachian tube play a key role in the development of ome in all ages. mechanical obstruction of the eustachian tube is one of the main identifiable causes of eustachian tube dysfunction, especially due to adenoid hypertrophy (ah) in the pediatric age. the presence of ah can obstruct the nasopharyngeal ostium of the eustachian tube, leading to negative pressure in the middle ear cavity, and eventually mucosal transudation [10] . indeed, ah is a frequent condition observed in patients with ome [7] [8] [9] 11] . in children with ah and ome, the surgical removal of the adenoid (associated with ventilation tube insertion) accelerates the recovery of the middle ear mucosa, decrease the risk of recurrence and need of repetitive surgical procedures, and reduce treatment failure rate. however, new evidence point out that the benefits of adenoidectomy in children with ome older than 4 years-old is independently of the adenoid size [12] , suggesting that the removal of a local reservoir of pathogenic microbiota (viruses, planktonic bacteria, and biofilms) is the key for such benefits [13] [14] [15] . despite some studies have evaluated the microbial colonization of adenoid and middle ear, no studies have thoroughly studied the association of respiratory pathogenic microbiota between these two niches. therefore, the present study was carried out to compare the detection of common respiratory viruses and bacteria in adenoids and middle ear fluid in children with ome and in controls. moreover, adenoid can be reservoirs of pathogens that may reach the middle ear [13] . adenoid samples obtained from patients with chronic adenoiditis are highly colonized by multiple species of viruses and bacteria. the rate of detection in hypertrophic adenoids is higher than 85% for viruses [14] and almost 100% for bacteria [15] . bacteria and viruses have been detected in the middle ear fluid (mef) from children with ome. using an rt-pcr-based assay only for three respiratory viruses (rhinovirus, respiratory syncytial virus and human coronavirus), positivity was detected in 30% of mef from children with ome in finland [16] . in a study conducted in pittsburgh, 75 of 97 (77.3%) samples of mef from children with ome were positive by pcr for one or more of three bacteria (m catarrhalis, h influenza and s pneumoniae) [17] . the study was conducted following the principles expressed in the declaration of helsinki, after approval by the ethics review committee of the clinical hospital of the university of sao paulo school of medicine in ribeirão preto, brazil (#10466/2008). written informed consent was obtained from all parents/guardians. the study enrolled patients seen at the division of otorhinolaryngology from may 2010 to august 2012. two groups of patients were studied: a) children with ome and adenoid hypertrophy (ah) who underwent adenoidectomy and tympanostomy with ventilation tube insertion, and b) children with no middle ear disease, who underwent cochlear implantation due to severe hearing loss. all patients were evaluated by otoscopy, audiometry, tympanometry, and nasal endoscopy. ome was diagnosed by medical history suggesting persistence of middle ear fluid for more than 3 months without acute signs of inflammation, confirmed by the presence of tympanic membrane opacity, retraction, or air-liquid interface, and by a type b tympanogram and/or conductive hearing loss. ah was defined at nasal endoscopy by the presence of adenoid in contact with the torus tubaris (grade 3) and/or vomer (grade 4) [18, 19] . exclusion criteria were: prior surgical procedures in the upper airways, including tympanostomy for tube placement; use of antibiotics or symptoms of respiratory infection within the last month; history of recurrent acute otitis media or recurrent upper airways infection; tympanic membrane perforation, or the presence of genetic syndromes, such as down syndrome. all patients with ome and ah were under treatment with nasal corticosteroids for at least one month during the preoperative period. the control group consisted of children undergoing cochlear implant due to severe hearing loss with no medical history of otitis media, no respiratory infection within the last month, absence of tympanometric alterations, nor adenotonsillar hypertrophy. all patients followed the vaccination protocols of the brazilian ministry of health [20] , which includes some pathogens targeted in the present study, such as pneumococcal conjugated. additionally, patients in the control group were immunized with pneumococcus 23-valent vaccine and meningococcus c, according to the cochlear implant surgery protocol. fragments of adenoid (surface and stroma) and middle ear washes (mew) were obtained from all patients under general anesthesia. briefly, adenoids fragments were collected using beckmann adenoid curette (from ome-ah patients) or punch biopsy forceps, under direct visualization after soft palate retraction, in order to avoid contamination (controls). adenoids samples were placed in eagle's minimal essential medium (mem) with 15% of a solution containing 20,000 u/ml of penicillin-streptomycin and 200 μg/ml of amphotericin b, 10% of fetal bovine serum (gibco 1 , life technologies, carlsbad, ca, usa), and kept on ice until further processing. adenoids were washed twice in mem to remove blood and tissue debris and the fragment was macerated in trizol 1 (invitrogen, life technologies, carlsbad, ca, usa) for later nucleic acid extraction. in children with ome, mew was performed with 0.5 ml of sterile saline through a small tympanostomy using a sterile needle coupled to a 5 ml syringe, after mechanical cleaning of the external ear canal. after that, the mew was conditioned into a polypropylene microtube and treated with antimycotic and antibiotic solution (gibco 1 , grand island, ny, usa) for one hour at 4˚c. aliquots were made in thrice the volume of trizol, and frozen at −80˚c until further processing. from each patient, samples were collected from only one side, which was randomly chosen when bilateral disease was present. in control patients, mew was obtained after partial mastoidectomy was performed, when direct access to the middle ear was possible through the attic, with the same sterile technique utilized to collect samples in ome patients. nucleic acid extraction and pathogen detection rna was extracted from 250 μl of mew or from approximately 30 mg of adenoid tissue using 750 μl of trizol 1 [21] , according to the manufacturer's protocol. dna was further extracted using dna purification kit (promega 1 , fitchburg, wi, usa) starting with the dna-enriched fraction obtained with trizol 1 . samples were tested by real-time pcr for rhinovirus (hrv), influenza virus (flu), parainfluenza virus (hpiv), syncytial respiratory virus (hrsv), metapneumovirus (hmpv), coronavirus (hcov), enterovirus (hev), adenovirus (hadv) and human bocavirus (hbov) following an in house real-time pcr protocol, based on primers listed on s1 table and taqman probes, with the same procedures published elsewhere (14] . real-time pcr was also used to detect the presence of s. pneumoniae, h. influenzae, m. catarrhalis, p. aeruginosa, and s. aureus, using the primers listed on s2 table. the fisher's exact test was used to compare rates of virus and bacteria detection in both groups of patients. wilcoxon test was done to assess correlation between bacteria and virus detected in each patient. the analysis was performed using graphpad prism 5 (la jolla, ca, usa). 14 control patients (2-12 years-old; mean 4 years 6 months; sd 2.64; 9 male / 5 female) and 37 children with ome and ah (2-12 years-old; mean 6 years 1 month; sd 1,97; 19 male / 18 female) were included in this study. no severe complications related to the sampling procedure, such as nasal or oral bleeding, dysphagia and otorrhea, were observed in the control group as well as in the ome-ah group within the first 48 hours after surgery or after 3 weeks postoperative reassessment. at least one virus species was detected, considering adenoid samples, in 32 of 37 (86.5%) patients with ome-ah and in 11 of 14 (78.6%) control patients. differently, at least one virus was detected in mews from 19 of 37 (51.3%) patients with ome, and in 3 of 14 (21.4%) control patients. the overall rates of virus detection were significantly higher in adenoids as compared to mew in both groups (p = 0.002 for the ome group and p = 0.007 for the control group), but the virus detection rates in mew and adenoids were not significantly different between patients with ome and control children (respectively p = 0.06 and p = 0.66). despite this fact, hadv was more often found in adenoids of ome-ah group as compared to controls (p = 0.01) ( table 1) . the rates of virus co-detection in positive samples were 59.3% (19/32) in adenoids and 21% (4/19) in mews. two or 3 viruses were simultaneously detected in adenoid tissues from 15 (40.5%) and 4 (10.8%) patients with ome. in contrast, only 3 (8.1%) control patients had 2 viruses and only 1 (2.7%) had 3 different viruses detected in mews (fig 1) . there was no significant concordance between detection of viruses in adenoid and the middle ear (p = 0.65). only 6 (16.2%) patients with ome (5 hev and 1 hbov) and 1 control patient (hev) had the same virus detected in mews and adenoid tissue, suggesting that the viral colonization of the middle ear is independent from viral infection of adenoids. notably, the rates of bacteria detection in mews was significantly higher in patients with ome than in control patients (p = 0.01), especially due to s. pneumococcus and m. catarrhalis. overall bacterial detection rates in adenoids were similar between ome and control patients (p = 0.29). twelve of 14 control patients (85.7%) had at least one species of respiratory pathogenic bacteria detected in adenoid tissues, reinforcing previous data that indicate that potentially pathogenic bacteria colonize adenoids of healthy children [22, 23] (table 2) . with regard to bacteria co-detection in adenoids and mews from ome patients, 18 patients (48.6%) with ome had more than one bacterium detected in adenoid, and two, three, or four bacteria were simultaneously detected respectively in 16.2%, 18.9%, and 13.5% of patients. six patients had 2 (16.2%), four had 3 (10.8%) and two patients had 4 bacteria (5.4%) simultaneously detected in mews for an overall frequency co-detection of bacteria in mews of 32.4% (12 of 37 patients). bacteria co-detection was also frequent in the control group, in which five patients had 2, three patients had 3 and one patient had 4 different bacteria simultaneously detected in adenoid tissues. bacteria co-detection was not observed in mews from control patients (fig 2) . similar to the findings regarding viruses, there was no concordance between bacteria detected in adenoids and mews, both in controls and ome patients (p = 0.18). the same bacteria were detected in the mews and adenoids was observed in 13 (35.1%) patients with ome, and in 2 (14.2%) controls (fig 2) . multiple associations between viruses and bacteria in the adenoid and middle ear were investigated. the only microbes that presented positive association was s. pneumoniae and hadv in hypertrophic adenoids of patients with ome (p = 0.02) (tables 3 and 4) . we did not find any significant association between virus and bacteria in the adenoid or middle ear in patients of the control group. although the pathogenesis of ome is not fully understood, there is evidence that adenoid may play an important role in this disease, either by mechanically impairing the eustachian tube function, or by acting as a microbial reservoir for ascending infection to the middle ear [13, 14, 24] . children are often exposed to pathogens that may chronically persist in tissues in the upper airways, especially in the adenoid and tonsils [13, 14, 25, 26] . thus, as the adenoid has an intimate anatomical relation with the eustachian tube and ultimately to the middle ear, it is important to understand how the presence of microbes in the adenoid could lead to the development or persistence of ome in children. to the best of our knowledge, this is the first case-control study that compared adenoid and middle ear microbiota of ome patients with healthy children. in our study, we used a sensitive method to detect nucleic acid of a comprehensive panel of respiratory viruses and bacteria to compare the microbial colonization of adenoid and its correspondence in the middle ear in both ome children and controls. the overall detection rate of bacteria in the middle ear was significantly higher in patients with ome than in controls, but was similar for viruses. the higher overall frequency of bacteria detection in mews from ome patients was expected, since the accumulation of middle ear effusion favors growing of bacterial elements that may have been suctioned through the tube from the nasopharyngeal microbiota. moreover, these bacteria may play roles as chronic inflammatory stimuli to the mucosa of the middle ear and contribute to the dysfunction of eustachian tube. there was little or no concordance of the microbe detected in the adenoid and the middle ear, both in patients with ome as well as in controls. this finding is in agreement with a previous report that performed 16s rdna pyrosequencing of both niches, showing that the adenoid microbiota was more diverse and complex than of the middle ear in a child with ome [27] . as the adenoid is exposed to the nasal and oral microbial contents, differently from the enclosed cavity of the middle ear, this makes the adenoid more susceptible to be colonized by a higher load and more variety of microorganisms. it is generally accepted that the development of negative pressure within the middle ear cavity suctions microbial components of the nasopharyngeal microbiota but, at present, there is no enough data to rule out the establishment of a selected microbiome especial to the middle ear cavity. interestingly, adenovirus detection was more frequent in hypertrophic adenoid tissues from patients with ome than in normal adenoids from healthy controls. this is in agreement with a paper previously published by our group reporting high rates of adenovirus detection by pcr in hypertrophic adenoids [14] . it has not been clear whether adenovirus has any role in pathogenesis of adenoid hypertrophy, especially because this virus is also frequent in nasopharyngeal secretions from healthy individuals [28] . since some hadv species are more prone than others to have more prolonged shedding [29] , prospective longitudinal studies of viruses in patients with hypertrophic tonsils should include determination of adenovirus species to evaluate whether they are associated with chronic tonsillar hypertrophy. the observed association of hadv with s. pneumoniae in adenoids could lead to speculate that the epithelial damage induced by hadv could somehow favor the secondary local proliferation of s. pneumoniae [30] . some viruses, for instance influenza, are known to favor pneumococcal colonization of the upper airways by multiple mechanisms, including exposure of receptors for pneumococcal adherence and by providing nutrients sources for bacterial growth [31] . however, the simultaneous presence of hadv and s. pneumoniae in adenoid tissue could be independent observations, due to the high frequencies of both microorganisms in the adenoid. s. pneumoniae and m. catarrhalis were detected more often in middle ear washes from ome patients than controls. considering that s. pneumoniae and m. catarrhalis are the most frequent pathogens detected in acute otitis media [6] , and are able to ascend through the eustachian tube [32] , causing ciliary damage to the airway epithelium and disrupting mucociliary flow, this may result in conditions for persistence in the middle ear compartment [33] [34] [35] [36] . in some cases, especially when ome is not clearly related to a mechanical obstruction of the eustachian tube, the toynbee effect of negative pressure within the middle ear may help ascendance of microbes through the tube, leading to colonization of the middle ear, which may be pivotal in ome pathogenesis. m. catarrhalis has been widely studied because of its importance in middle ear diseases, and this interest has even led to vaccines for m. catarrhalis being proposed [37] . it's mechanisms of adherence and triggering of the immune response have been well studied and even when not related to clinically relevant infection processes, appears with a high level of colonization during childhood [38] . once regarded as nonpathogenic, m. catarrhalis could be related to several chronic affections of the upper airways [39] . microorganisms in sessile biofilms are frequently resistant to the innate and adaptive immune responses and to antimicrobial agents. in ome, there has been evidence that the presence of middle ear fluid is associated to formation of biofilm in the middle ear, and that these biofilms are related to the local inflammation [9, 24] . in conventional, culture-based microbiology, detection of these microorganisms is not always possible, and the use of sensitive molecular methods, such as real-time pcr, enables detection of multiple microbes in complex niches like the middle ear. although pcr cannot distinguish viable from dead microbes, the specificity of the primers coupled with careful sampling technique in order to minimize crosscontamination between sampling sites, ensure that the microorganisms were indeed present in the middle ear. viruses and bacteria interact in many different ways on different mucosal surfaces [40] , including the upper respiratory tract mucosa and tonsils [41] . persistence of viral infections includes a complete reprogramming of the local immune response, attenuation of production of type 1 interferon, and local alteration of the cd4+/cd8+ t cell balance [41, 42] . the airway mucosa affected by viral infection becomes more susceptible to bacterial adherence and mucosal inflammation [30, 43, 44] . therefore, associations of viruses and bacteria could be acting in concert to modify the course of the ome. in the present study, there was an association of detection of hadv and s. pneumoniae in adenoid tissues from patients with ome, suggesting that microbial correlations already described in acute middle ear disease [16, 45] could play a role in such chronic processes. clinical studies based on microbial detection in patients with adenoid hypertrophy and ome have rarely included a healthy control group. a notable example was a study of biofilms in middle ear samples, including adults and children [25] . importantly, biofilms were not observed in ome patients, but not in control specimens of middle ear mucosa obtained from patients undergoing cochlear implantation, strongly suggesting that biofilm production is an important factor in development of ome. through a minimally invasive method (mew) and a very sensitive procedure (real time pcr), we were able to compare samples of healthy children with those of children with ome. in the light of that result, the findings of some potentially pathogenic bacteria in the middle ear of patients undergoing cochlear implant suggests that their presence in the middle ear is independent of biofilm formation. unfortunately, in the present study no attempt was made to verify the presence of biofilm. concluding, in children with ome and adenoid hypertrophy we observed higher detection rates of potentially pathogenic bacteria, but not respiratory viruses, by real-time pcr in middle ear samples, as compared to control patients without adenoid hypertrophy. we did not observe correlation between the microbiota of adenoid and middle ear, neither in ome children nor in controls. this adds evidence that microbial community present in the middle ear may be associated with persistence of fluid and pathogenesis of ome. supporting information s1 conceptualization: gpb ea et wta-l fcpv. formal analysis: gpb. clinical practice guideline: otitis media with effusion clinical practice guideline: otitis media with effusion (update). otolaryngology-head and neck surgery burden of disease caused by otitis media: systematic review and global estimates chronic otitis media with effusion in infancy. how frequent is it? how does it develop? antibiotic therapy to prevent the development of asymptomatic middle ear effusion in children with acute otitis media: a meta-analysis of individual patient data otitis media the role of topical nasal steroids in the treatment of children with otitis media with effusion and/or adenoid hypertrophy increased risk of otitis media with effusion in allergic children presenting with adenoiditis association of adenoid surface biofilm formation and chronic otitis media with effusion laterally hypertrophic adenoids as a contributing factor in otitis media role of mometasone furoate aqueous nasal spray for management of adenoidal hypertrophy in children the protective effect of adenoidectomy on pediatric tympanostomy tube re-insertions: a population-based birth cohort study adenoid reservoir for pathogenic biofilm bacteria high rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease comparative study of bacterial strains and antibiotic susceptibility tests between chronic tonsillitis patients with iga nephropathy and without nephritis polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion molecular analysis of bacterial pathogens in otitis media with effusion assessment of adenoid size in children by fibreoptic examination validation of a new grading system for endoscopic examination of adenoid hypertrophy secretaria de vigilância em saúde. departamento de vigilância das doenças transmissíveis manual de normas e procedimentos para vacinação. brasília: ministério da saúde a reagent for the single-step simultaneous isolation of rna, dna and proteins from cell and tissue samples differences in nasopharyngeal bacterial carriage in preschool children from different socio-economic origins differences in nasopharyngeal bacterial flora in children with nonsevere recurrent acute otitis media and chronic otitis media with effusion: implications for management role of adenoid biofilm in chronic otitis media with effusion in children direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media respiratory viruses are continuously detected in children with chronic tonsillitis throughout the year the otologic microbiome: a study of the bacterial microbiota in a pediatric patient with chronic serous otitis media using 16srrna gene-based pyrosequencing otitis media with effusion a comparison of viral fitness and virulence between emergent adenovirus 14p1 and prototype adenovirus 14p strains the airway epithelium: soldier in the fight against respiratory viruses influenza promotes pneumococcal growth during coinfection by providing host sialylated substrates as a nutrient source genetic similarity between adenoid tissue and middle ear fluid isolates of streptococcus pneumoniae, haemophilus influenzae and moraxella catarrhalis from iranian children with otitis media with effusion secretory otitis media and the nature of the mucociliaryy system otitis media with effusion: components which contribute to the viscous properties viral rna in middle ear mucosa and exudates in patients with chronic otitis media with effusion mucociliary clearance defects in a murine in vitro model of pneumococcal airway infection moraxella catarrhalis: from emerging to established pathogen moraxella catarrhalis, a human respiratory tract pathogen moraxella catarrhalis: pathogenic significance in respiratory tract infections treated by community practitioners viruses and the microbiota distinct regulation of tonsillar immune response in virus infection innate and adaptive immune regulation during chronic viral infections mucosal exudation of fibrinogen in coronavirus-induced common colds respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell typedependent manner natural history of untreated otitis media key: cord-298941-xf2ukinp authors: al-abdallat, mohammad mousa; payne, daniel c.; alqasrawi, sultan; rha, brian; tohme, rania a.; abedi, glen r.; nsour, mohannad al; iblan, ibrahim; jarour, najwa; farag, noha h.; haddadin, aktham; al-sanouri, tarek; tamin, azaibi; harcourt, jennifer l.; kuhar, david t.; swerdlow, david l.; erdman, dean d.; pallansch, mark a.; haynes, lia m.; gerber, susan i. title: hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description date: 2014-05-14 journal: clinical infectious diseases doi: 10.1093/cid/ciu359 sha: doc_id: 298941 cord_uid: xf2ukinp background: in april 2012, the jordan ministry of health investigated an outbreak of lower respiratory illnesses at a hospital in jordan; 2 fatal cases were retrospectively confirmed by real-time reverse transcription polymerase chain reaction (rrt-pcr) to be the first detected cases of middle east respiratory syndrome (mers-cov). methods: epidemiologic and clinical characteristics of selected potential cases were assessed through serum blood specimens, medical record reviews, and interviews with surviving outbreak members, household contacts, and healthcare personnel. cases of mers-cov infection were identified using 3 us centers for disease control and prevention serologic tests for detection of anti–mers-cov antibodies. results: specimens and interviews were obtained from 124 subjects. seven previously unconfirmed individuals tested positive for anti–mers-cov antibodies by at least 2 of 3 serologic tests, in addition to 2 fatal cases identified by rrt-pcr. the case-fatality rate among the 9 total cases was 22%. six subjects were healthcare workers at the outbreak hospital, yielding an attack rate of 10% among potentially exposed outbreak hospital personnel. there was no evidence of mers-cov transmission at 2 transfer hospitals having acceptable infection control practices. conclusions: novel serologic tests allowed for the detection of otherwise unrecognized cases of mers-cov infection among contacts in a jordanian hospital-associated respiratory illness outbreak in april 2012, resulting in a total of 9 test-positive cases. serologic results suggest that further spread of this outbreak to transfer hospitals did not occur. most subjects had no major, underlying medical conditions; none were on hemodialysis. our observed case-fatality rate was lower than has been reported from outbreaks elsewhere. in april 2012, the jordan ministry of health (jmoh) investigated a cluster of 13 suspected pneumonia cases among healthcare personnel, of which 2 were fatal, at a hospital in the city of zarqa [1] . despite testing for multiple potential pathogens, the investigation did not identify a known etiology for these infections. following the discovery of middle east respiratory syndrome coronavirus (mers-cov) in september 2012 [2] , specimens from the 2 fatal cases in jordan were retrospectively tested and both yielded positive results for mers-cov by real-time reverse transcription polymerase chain reaction (rrt-pcr), and were reported to the world health organization (who). these were the first confirmed human cases of infection with this emergent virus, which continues to appear as sporadic cases and clusters internationally, and which is now the focus of worldwide public health investigation and response [3, 4] . using newly developed serologic assays to determine mers-cov antibody responses among case contacts in this outbreak, epidemiologists from the jmoh, us centers for disease control and prevention (cdc), and regional partners conducted a retrospective seroepidemiologic investigation to (1) confirm whether surviving outbreak members had presence of antibodies to mers-cov, (2) ascertain whether viral transmission occurred among household contacts or to other healthcare personnel, and (3) describe the clinical features of all detected mers-cov infections in jordan. we interviewed and collected serum specimens from available members of the initial outbreak (who were admitted to the focal outbreak hospital during the period from 15 march to 30 april 2012 with fever and dry cough, and with radiological evidence of pneumonia), their household contacts (who reported usually sleeping under the same roof as a defined outbreak member during february-april 2012), a sample of healthcare personnel from 3 medical institutions that admitted outbreak subjects (nonsystematic enrollment, with preference toward those reporting close contact with outbreak members), and field investigators from the jmoh. hospitalized subjects meeting the initial outbreak case definition were subsequently transferred from the focal outbreak hospital to 2 other hospitals in amman. participating healthcare personnel were employed at one of these hospitals or at jmoh during february-april 2012. epidemiologic data were obtained through medical record reviews and personal interviews during our may 2013 investigation. interviews were conducted in arabic, and documented contact history (with outbreak members, household members, visiting travelers, and animals) and occupational exposures. we conducted medical record reviews and key informant interviews with clinicians who provided medical care to patients with suspected infection and heads of infection control units at each medical institution and at the jmoh. informed consent was obtained prior to serum collection and interviews. as a public health response to a disease outbreak, this investigation did not require institutional review board review. all work with live mers-cov was done in cdc biosafety level 3 (bsl-3) containment facilities in atlanta, georgia. serum samples were inactivated using 2 × 10 6 rads γ-irradiation and stored at −80°c until use. to maximize specificity, we defined mers-cov antibody positivity as subjects having correlated, positive laboratory results from the hku5.2n screening enzyme-linked immunosorbent assay (elisa), as well as confirmed positive results by either the mers-cov immunofluorescence assay (ifa) or the mers-cov microneutralization assay (mnt) (supplementary table 1 ). an initial indeterminate test result was recorded for those subjects having only a single, uncorrelated positive test result. antibody detection by hku5.2 nucleocapsid elisa and mers-cov ifa and mnt genetic sequencing data indicate that mers-cov is a β-coronavirus (subgroup 2c) similar to the bat covs hku4 and hku5. the recombinant bthku5.2 nucleocapsid proteinbased elisa was developed by the cdc to detect the presence of antibodies that cross-react with the hku5.2 n protein in serum samples from possible mers cases. if cross-reactive antibodies were detected in serum samples, then confirmation of mers-specific antibodies was determined by either mers-cov mnt or ifa. pi-batcov hku5.2 nucleocapsid (n) gene in pet-28b (+) plasmid was provided by dr susanna lau, university of hong kong. his-tagged recombinant protein was expressed in escherichia coli and purified by metal affinity chromatography. recombinant hku5.2n protein indirect elisa was developed using a modified version of the severe acute respiratory syndrome (sars) cov n elisa described by haynes et al [5] . sera were considered positive when the optical density (od) values were at or above the 0.43 cutoff value (mean absorbance at 405 nm of sera from us blood donors plus 3 standard deviations). the overall specificity of the assay was determined after screening 545 serum samples from donors in the united states and the middle east and persons with other non-mers respiratory infections (eg, human coronavirus [hcov] oc43, hcov-229e, sars-cov, hcov-nl63, rhinovirus, human metapneumovirus, h1n1). the assay specificity was 96.7% (527/545). serum from hku1 human serum was not available for evaluation; however, hku1 mouse hyperimmune serum did not cross-react with the hku5.2 n protein. at a screening dilution of 1:400, sera with od values at or near the cutoff were titered with serial 4-fold dilutions (1:100-1:6400) and further evaluated using mers-cov (hu/jordan-n3/2012) (genbank kc776174.1) ifa and mnt. indirect immunofluorescence was performed by screening sera at a dilution of 1:50 or 1:100 on paraformaldehyde-fixed, acetone-methanol-permeabilized, mers-cov-infected oruninfected control vero cells. the source of the positive control for this assay was a serum sample from a patient infected with mers-cov hu/england-n1/2012 ( provided by m. zambon, public health england). antihuman immunoglobulin (ig) g, igm, and iga fluorescein isothiocyanate conjugate was used and specific fluorescence was detected under an immunofluorescence microscope. a positive result was scored when fluorescent intensity equaled or was higher than that of the positive control. a weakly positive result was scored when fluorescent intensity was lower than that of the positive control. serum samples were tested for the presence of neutralizing antibodies to mers-cov using a modified mnt method described for sars-cov [6] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell monolayer lysis in at least 1 of the 3 triplicate wells. controls were included for each mnt assay performed, including the input virus back titration and mock-infected cells. all assay results were confirmed in 3 separate assays, and representative data are presented. tests of statistical significance were performed between the mers-cov antibody-positive and -negative subjects, including fisher exact test and χ 2 tests for categorical variables using sas software version 9.3 (sas institute, cary, north carolina). serologic specimens and interviews were obtained from 124 subjects. we obtained serologic specimens and data from 9 of the 11 (82%) surviving members meeting the initial outbreak case definition; the remaining 2 subjects were unable to be interviewed (1 member was lost to follow-up and 1 did not consent) ( figure 1 ). we also enrolled 26 household contacts and 89 subjects who did not meet the initial outbreak case definition who worked in healthcare and allied professions. among the healthcare personnel interviewed, 58% were nurses, 21% were physicians, and the remaining were allied health professionals; approximately half were employed at the focal outbreak hospital. seven of the 124 subjects tested positive for anti-mers-cov antibodies by both hku5.2 elisa and ifa (table 1 and supplementary figure 1) , and all but 1 also had detectable neutralizing antibody titers as determined by mnt. the subject who did not have detectable neutralizing antibodies was testpositive both by hku5.2n elisa and by a confirmative ifa. demographic and epidemiologic comparisons of seropositive and seronegative subjects are provided in supplementary table 2 . sera from the 2 fatal cases (designated outbreak subjects 01 and 12) having positive rrt-pcr tests were also tested by the 3 described serology tests. a serum sample from outbreak subject 01 (taken 16 days after onset of respiratory symptoms) was positive by hku5.2n elisa and ifa and had detectable mers-cov neutralizing antibodies. two serum specimens from outbreak subject 12 (collected 26 and 32 days after onset) were negative for anti-mers-cov antibodies. of the 7 subjects found to be positive for anti-mers-cov antibodies during this investigation, 6 were surviving members of the initial outbreak group and 1 was previously unrecognized. thus, including the 2 fatal cases previously detected and reported, a total of 9 individuals in this outbreak had evidence of mers-cov infections by acute rrt-pcr tests (n = 2) or convalescent antibody tests (n = 7). the case-fatality rate among all test-positive subjects was 22% (2 of 9). we documented that each serologic test-positive subject had unprotected mers-cov exposure(s) to at least 1 rrt-pcr test-positive subject. an additional 8 subjects had single positive test results by either hku5.2n elisa or ifa, but their mers-cov antibody status was considered indeterminate because both tests were not positive (table 1) . we obtained specimens and data from a total of 97 healthcare personnel who worked during february-april, 2012, representing a majority of intensive care (intensive care unit [icu] and coronary care unit [ccu]) personnel at the outbreak hospital as well as other personnel having close contact with initial outbreak investigation members ( figure 1 ). these included 8 surviving outbreak members who were healthcare personnel at the focal outbreak hospital and were not lost to follow-up, 49 other personnel at the focal outbreak hospital, 16 personnel at transfer hospital a, 20 personnel at transfer hospital b, and jmoh's 4 outbreak investigators. of the 57 healthcare personnel at the focal outbreak hospital who survived and the 1 who died, 6 (10%) had cases of mers-cov. our investigation provided no evidence of mers-cov infections or transmission events among personnel at the 2 receiving transfer hospitals, even though some patients were transferred temporally close to their symptom onset dates. interviews with surviving subjects and family members revealed that transmission opportunities among healthcare personnel were not restricted to the workplace. we obtained serologic specimens from members of 11 households, including those from the initial outbreak group and another 26 subjects who had resided in those outbreak member abbreviations: elisa, enzyme-linked immunosorbent assay; ifa, immunofluorescence assay; mers-cov, middle east respiratory syndrome coronavirus; mnt, microneutralization titer. a outbreak member 08 was lost to follow-up, and outbreak member 13 did not consent. outbreak members 01 and 12 were previously laboratory-confirmed positive by real-time reverse transcription polymerase chain reaction (rrt-pcr) and died. serum samples from outbreak members 01 and 12 were collected prior to death and stored. b serum specimens with optical density (od) values ≥0.43 at a 1:400 dilution against hku5.2n elisa were considered to be positive. specimens were further titered against hku5.2n at 1:100, 1:400, 1:1600, and 1:6400 dilutions. the antibody titer was taken to be the highest antibody dilution above the cutoff od that yielded a ratio of the absorbance of the positive serum and negative serum (p/n) > 3. the value is the reciprocal of the dilution. c serum specimens that were positive by hku5.2n elisa were screened at either 1:50 or 1:100 by indirect ifa using mers-cov_jordan-infected vero cells. h outbreak members conformed to the original outbreak definition; however, some were retrospectively determined to be mers-cov test negative. they were part of the original, defined outbreak that our investigation used to trace a priori contacts and exposures, so this descriptive title is retained. i hku5.2 n elisa od values for serum specimens from outbreak members 05, 07, and 10 and from healthcare personnel 308 were near the assay cutoff od value and rescreened by serial dilution. these serum samples were initially weakly positive by ifa and considered initially indeterminate. upon rescreen by ifa, the samples were determined to be negative for the presence of mers-cov antibodies. j although outbreak member 12 was positive for mers-cov by rrt-pcr, his sera were antibody negative. presumably, this subject died before an antibody response was detectable. this case is considered to be confirmed by current who mers-cov diagnostic guidelines. households during the outbreak period. one household was lost to follow-up, and 1 did not consent for participation. from one of these households was the symptomatic wife of an initial outbreak investigation member who tested positive for mers-cov antibodies. twelve household subjects were children <18 years old, all of whom were serologically test negative. a summary of underlying conditions for test-positive subjects, including the 2 fatal cases initially identified by rrt-pcr (outbreak members 01 and 12), is presented in table 2 . of the 9 testpositive subjects, 66% were male, with a median age of 40 years (range, 25-60 years) at illness onset. we found no evidence of underlying immunodeficiency or immunosuppressant medications/therapies among any of these subjects. one subject had an atrial septal defect, 2 had a history of hypertension, 2 were smokers at the time of illness, and 1 reported a pregnancy of 5 months' gestation. although diabetes mellitus has been observed as a potential risk factor for mers-cov [7] , none of the subjects reported here had a prior diagnosis of diabetes mellitus and, based on serum glucose values taken during their hospitalizations, none had indications of undiagnosed diabetes mellitus. the most common presenting symptoms, as documented in medical charts, included fever (89%), cough (89%), dyspnea (56%), chest pain (44%), and malaise (33%). eight subjects presented for hospital care a median of 5 days after symptom onset (range, 1-14 days). of these patients, 7 (88%) had cough, 7 (88%) had documented fever (temperature (≥38.0°c), 6 (75%) had dyspnea, 5 (63%) had chest pain, and 5 (63%) had malaise at some point during their disease course. less common symptoms included chills (38%), wheezing (25%), and diarrhea, vomiting, sore throat, palpitations, and confusion (13% each). seven subjects had abnormal chest radiographic findings reported within 3 days of presentation, and 3 of those 7 had bilateral findings. of the remaining 4 subjects with initial unilateral findings, 3 went on to develop bilateral infiltrates later in their hospitalization, documented either by chest radiography or computed tomography (ct). one subject (outbreak member 12) received an initial diagnosis of pericarditis, and a ct scan with abnormal pulmonary findings was reported 4 days later ( table 3) . seven of the 8 subjects (88%) who presented to medical care were admitted; 1 refused admission. six subjects (75%) required respiratory support with at least supplemental oxygen, and 4 subjects (50%) received intensive care (in ccu or icu), but only the 2 (25%) patients who died required mechanical ventilation, of which 1 patient also required pressor support (dopamine and norepinephrine) for cardiorespiratory failure. complications among hospitalized subjects were also limited to the 2 patients who died, 1 of whom had hyperkalemia with associated ventricular tachycardia, disseminated intravascular coagulation, and eventual cardiac arrest; the other had pericarditis, pericardial and pleural effusions, and supraventricular tachycardia late in the course of illness. although leukopenia (<4.0 × 10 9 /l) was observed in 2 subjects, lymphopenia (<1.5 × 10 9 /l) was observed in 6 of the 7 subjects who had documented complete blood counts with differentials (86%). elevated leukocyte counts (>11 × 10 9 /l) were observed during the course for 2 subjects (25%), both of whom died. these 2 subjects also had laboratory abnormalities consistent with multiorgan system failure late in the course of disease. these included evidence of elevated alanine aminotransferase and aspartate aminotransferase (>40 u/l) and significant coagulopathy with an international normalized ratio of >1.5, as well as thrombocytopenia (<140 × 10 9 /l). in addition, the 2 subjects who died had elevated serum creatinine measurements (≥133 µmol/l) on the day of their deaths. a third case had an isolated elevated creatinine measurement, but had a subsequent normal value the following day. no patient received hemodialysis (table 3) . outbreak member 01 died 17 days after onset of symptoms (on day 11 of hospitalization) and outbreak member 12 died 35 days after onset of symptoms (on day 22 of hospitalization). the remaining 7 subjects survived, and the 5 who were hospitalized were discharged following a median of 8 days (range, 4-16 days). despite having respiratory symptoms, the pregnant household subject did not seek medical care due to concerns regarding receiving chest radiography and medications. this pregnancy resulted in stillbirth during the course of her illness [8] . surviving subjects and the family members of deceased patients reported that contact with animals was rare in this urbanized area, and no contact with camels was identified among subjects having early symptom onsets. furthermore, none of the subjects had traveled to, or had received visitors from, the arabian peninsula shortly prior to symptom onset. at the focal outbreak hospital, there were no physical barriers between ccu and icu beds, spaced approximately 3 meters, with the exception of cloth drapes in the ccu. isolation or negativepressure rooms were not present, and infection control compliance issues were reported during the outbreak. infection control insufficiencies were not noted at the 2 receiving transfer hospitals. we used novel serologic assays to determine antibody responses of subjects from a mers-cov outbreak investigation in jordan, including the earliest cases of this emerging virus yet discovered. in addition to 2 fatal cases confirmed by rrt-pcr and reported to who, we discovered 7 previously unconfirmed and unreported mers-cov infections. detection of these 7 additional antibody-positive subjects, including healthcare personnel from the focal outbreak hospital and a family contact of 1 antibody-positive subject, and the establishment of contacts with mers-cov infected subjects when potentially infectious, suggests that human-to-human transmission of mers-cov occurred. although community exposures were possible, healthcare-associated transmission was a plausible explanation for healthcare personnel infections. mers-cov infections were not detected among healthcare personnel at a transfer hospital having better adherence to infection control measures. compared with published descriptions of saudi arabian and french cases [9] [10] [11] [12] , among the 9 total jordanian cases identified through our collaborative investigation, subjects were younger and had fewer underlying medical conditions, and there was a lower case-fatality rate. although all subjects with mers-cov infection in our investigation had acute respiratory illnesses during the outbreak period, 78% of those who were infected survived. most subjects had no underlying medical conditions and none were on hemodialysis or had indications of diabetes mellitus. one newly detected subject, who was a household contact, did not seek medical care. our data support the probability that, in outbreak settings, infections may remain undetected among subjects who have mild symptoms, lack predisposing conditions, or have barriers to accessing appropriate diagnostic care. therefore, the true mers-cov case-fatality rate may be lower than that based on symptomatic, hospitalized cases alone. the presenting symptoms we observed were largely consistent with those of previously described mers-cov cases [13] [14] [15] [16] and included fever with respiratory symptoms such as cough and dyspnea, and associated infiltrates on chest radiography. on initial presentation, many subjects did not have evidence of bilateral pneumonia. although gastrointestinal symptoms such as vomiting and diarrhea were documented for 2 subjects, we did not observe these as presenting symptoms, as they were in saudi arabian and french cases. once hospitalized, lymphopenia, a prominent laboratory feature among previously described cases, was observed in the majority of our subjects. however, other laboratory abnormalities observed in previous reports, such as thrombocytopenia, were limited mostly to the 2 fatal cases late in the course of illness, consistent with multiorgan system failure. also, unlike previously reported cases, renal failure was not a prominent clinical feature among our subjects, as renal dysfunction was observed only in the 2 fatal cases on the day of death. rapid isolation of patients with suspected mers-cov and rigorous infection control practices at the receiving transfer hospitals may have been important in preventing transmission at these locations. hospitals should have established policies and procedures for the rapid identification of suspected or known mers-cov cases and implementation of appropriate infection prevention measures. the cdc recommends standard, contact, and airborne precautions for the management of hospitalized patients with known or suspected mers-cov infection [17] . one jordanian patient was initially hospitalized with pericarditis, a manifestation similar to 1 mers-cov case occurring in the kingdom of saudi arabia [9] . although this jordanian patient's serologic specimens tested negative for mers-cov antibodies at periods throughout his hospital stay, 1 acute specimen collected several days before death was confirmed positive for the virus by rrt-pcr. these laboratory findings and the patient's exposure in the ccu, where he was situated in the bed directly next to another patient with rrt-pcr-confirmed mers-cov, collectively suggest the likelihood that the patient was nosocomially infected with mers-cov and died before an antibody response was detectable. based on the knowledge of sars-cov antibody responses, igg and neutralizing antibodies to sars-cov peaked 4 months following a patient's recovery from acute infection [18] . antibody levels did decline over time, but detectable sars-cov neutralizing antibodies persisted up to 2 years after onset of sars-cov symptoms [19, 20] . approximately 13 months had passed between our may 2013 investigation and the april 2012 outbreak. although this was sufficient time for infected subjects to produce an antibody response to mers-cov, the role of waning immunity on the antibody response [21] and whether persistence of these antibodies is important for protection from reinfection remain unclear. we implemented a rigorous case definition based on an elisa-positive result plus at least 1 correlating assay result to maximize specificity. infections with sars-cov triggered humoral and cellular immune responses in all studied humans [22] , and high titers of neutralizing antibodies were observed in response to sars-cov infections, but such characteristics of the mers-cov immunologic response remain unknown. as for those indeterminate laboratory findings among subjects with documented mers-cov exposure(s) but having only an elisa-positive result and mild or absent respiratory symptoms, it is possible that the viral exposure to these subjects did not trigger a long-lasting ifa-or mnt-recognizable immune response. because obtaining appropriate lower respiratory specimens from subjects having mild or asymptomatic infections is challenging, the use of serologic assays to identify otherwise undetected cases of mers-cov has been demonstrated to be a useful tool. serological surveys have been conducted in retrospective case investigations around instances of mers-cov importations in europe [23] , as well as for establishing estimates of mers-cov seroprevalence among populations at risk [24] . further validation of serologic assays and assessments of how they complement rrt-pcr testing is needed. our investigation was unable to find evidence of any exposure (either zoonotic contacts, human contacts from the arabian peninsula, or among hospitalized contacts preceding the earliest symptomatic cases) that might explain the origin of the virus. the precise route(s) of mers-cov transmission remains unclear overall, but several mers-cov sequences have been identified in dromedary camel nasal secretions, including one that is indistinguishable from that found in infected humans [25] . in conclusion, the jordan respiratory illness outbreak in april 2012 resulted in a total of 9 test-positive mers-cov subjects. the source of the virus in these earliest known mers-cov cases remains unknown. compared with other reports, the improved survivability we observed is perhaps related to the youth and relative lack of underlying illnesses among the subjects we investigated. infection control practices at both transfer receiving hospitals may have been important in preventing mers-cov transmission in those facilities. since the discovery of the mers-cov, enhanced surveillance for severe acute respiratory illnesses in jordan has been implemented. international severe acute respiratory infection surveillance, collaborative investigations, and vigilance among healthcare providers are necessary components for addressing and preventing the further spread of mers-cov worldwide. supplementary materials are available at clinical infectious diseases online (http://cid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. epidemiological findings from a retrospective investigation isolation of a novel coronavirus from a man with pneumonia in saudi arabia update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide global alert and response (gar): novel coronavirus infection-update (middle east respiratory syndrome coronavirus) recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association middle east respiratory syndrome coronavirus (mers-cov): a case-controlled study of hospitalized patients stillbirth during infection with middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission epidemiological, demographic and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus a patient with severe respiratory failure caused by novel human coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection severe respiratory illness caused by a novel coronavirus family cluster of middle east respiratory syndrome coronavirus infections interim infection and prevention control recommendations for hospitalized patients with middle eastern respiratory syndrome coronavirus (mers-cov) disappearance of antibodies to sars-associated coronavirus after recovery two year prospective study of the humoral immune response of patients with severe acute respiratory syndrome longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance neutralizing antibodies in patients with severe acute respiratory syndrome associated coronavirus infection sars immunity and vaccination contact investigation for imported case of middle east respiratory syndrome lack of mers coronavirus neutralizing antibodies in humans middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia author contributions. d. c. p. had full access to all the data in the study and had final responsibility for the decision to submit for publication.disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.financial support. this work was supported by the us global disease detection operations center outbreak response contingency fund.potential conflicts of interest. all authors: no potential conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-349643-jtx7ni9b authors: uyeki, timothy m.; erlandson, karl j.; korch, george; o’hara, michael; wathen, michael; hu-primmer, jean; hojvat, sally; stemmy, erik j.; donabedian, armen title: development of medical countermeasures to middle east respiratory syndrome coronavirus date: 2016-07-17 journal: emerg infect dis doi: 10.3201/eid2207.160022 sha: doc_id: 349643 cord_uid: jtx7ni9b preclinical development of and research on potential middle east respiratory syndrome coronavirus (mers-cov) medical countermeasures remain preliminary; advancements are needed before most countermeasures are ready to be tested in human clinical trials. research priorities include standardization of animal models and virus stocks for studying disease pathogenesis and efficacy of medical countermeasures; development of mers-cov diagnostics; improved access to nonhuman primates to support preclinical research; studies to better understand and control mers-cov disease, including vaccination studies in camels; and development of a standardized clinical trial protocol. partnering with clinical trial networks in affected countries to evaluate safety and efficacy of investigational therapeutics will strengthen efforts to identify successful medical countermeasures. f rom september 2012 through april 27, 2016, a total of 1,728 laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) infections, leading to 624 deaths (36% case-fatality proportion), had been reported to the world health organization (who) (1) . most infections (75%) have been identified in saudi arabia (2) . zoonotic transmission from exposure to mers-cov-infected arabian camels, known as dromedaries, or their raw milk and limited, nonsustained human-to-human transmission have been reported, including large outbreaks in healthcare facilities (3) (4) (5) . the recovery of infectious mers-cov in virus cultures of specimens from bed sheets, bedrails, intravenous fluid hangers, and radiograph equipment indicates the potential for fomite transmission of the virus in hospitals providing care for mers-cov patients (6) . however, sustained human-to-human transmission has not been documented, and some case-patients have no identified source of exposure to mers-cov. as of april 2016, a total of 26 countries had reported locally acquired or exported cases from the arabian peninsula, including 2 cases in the united states identified during may 2014 in healthcare personnel who became ill after working in saudi arabia (7, 8) . a traveler who visited saudi arabia, qatar, the united arab emirates, and bahrain and then returned to south korea infected with mers-cov in mid-2015 triggered 184 mers-cov cases, resulting in 38 deaths in multiple health facilities and 1 additional case in a person who traveled to china (9, 10) . human infections with mers-cov are expected to continue to occur on the arabian peninsula because of the prevalence of mers-cov in dromedaries and the cultural importance of these camels (i.e., for food, milk, and racing purposes) in the region. during the 2003 outbreak of severe acute respiratory syndrome (sars) in china, civet cats, the suspected reservoir of sars coronavirus (sars-cov), were culled aggressively; no outbreaks were identified after 2004. in contrast, culling of camels is culturally impractical in the middle east, and mers-cov zoonotic infections of humans have continued since 2012. the potential for emergence of mers-cov mutations that could facilitate sustained community transmission and global dissemination cannot be predicted. no vaccines against or specific treatments for human infection with sars-cov, mers-cov, or other coronaviruses have been approved. since 2013, efforts have focused on furthering development of animal models, vaccines, and therapies against mers-cov (11,12). in this report, we update the current state of development for mers-cov medical countermeasures, including regulatory challenges in the united states, and draw attention to areas in immediate need of increased infrastructure support for development of these countermeasures. persons at higher risk for more severe disease, including persons >65 years of age and those with chronic medical conditions. therapeutic drugs with specific activity against mers-cov (e.g., antiviral drugs, immunotherapeutic treatments) or that target the host immune response could be used for treatment of human illness caused by mers-cov infection or for pre-or postexposure prophylaxis. before human clinical trials of potential mers-cov medical countermeasures are started, proof-of-concept data must be obtained from in vivo studies of experimentally infected animals. such data may indicate a product's potential efficacy and provide a mechanism for selection of available medical countermeasure candidates. in addition, mers-cov vaccines could be developed for animals and used for vaccination of dromedaries on the arabian peninsula and in source countries for camel imports to the horn of africa to reduce mers-cov transmission among camels and possibly from camels to humans. preclinical development of mers-cov medical countermeasures has been hindered by several factors, including limited data on the natural history of mers-cov infection in humans; the lack of a small animal model that is naturally susceptible to mers-cov; and the inability to consistently replicate severe human disease in mers-cov-infected nonhuman primates (nhps). another factor is limited access to clinical samples and recent virus isolates; for example, a mers-cov strain isolated from a patient in 2012, rather than a more recently isolated strain, is currently used by most investigators worldwide. small animal and nhp models are useful for testing potential medical countermeasures for efficacy (table 1) . studies in mice, both dipeptidyl peptidase-4 (dpp4 or cluster differentiation 26) transduced and transgenic, and in rabbits, hamsters, and ferrets have been reviewed elsewhere (16, 20, 21) . these small animal models have been used for screening potential mers-cov medical countermeasures (13, 14, 22) . the major nhp models under development include rhesus macaques and common marmosets (17, 18, 23) . overall, common marmosets appear to be better suited than rhesus macaques for therapeutic studies designed to target severe disease because marmosets show slightly slower onset of illness and longer duration and severity of disease and their small size requires lower doses of therapeutic drugs. however, the marmoset model has not been standardized and is not consistent between laboratories (18, 24, 25) . furthermore, the size of marmosets substantially limits sequential blood sampling for virologic or pharmacokinetic testing. challenges to the development of nhp models include determination and standardization of the optimal mers-cov challenge dose and of the volume and route of exposure, as well as the limited availability of nhps, especially marmosets. large animal models in development include camels and camelids such as alpacas (19, 26, 27) . these models may be vital in understanding the virology and immunology of mers-cov infection in dromedaries, a natural host. in addition, serologic evidence of mers-cov infection in alpacas has been reported in qatar (28) . major gaps for all animal models include a lack of consensus and availability of the optimal animal model to replicate severe human illness from mers-cov infection; limited availability of currently or recently circulating mers-cov strains; the lack of understanding of clinically relevant symptoms that can be incorporated into clinical scores or used as a signal to begin treatment in animal models; and competition for funding, laboratory space, availability of animals, and expertise with other emerging or reemerging infectious diseases, such as ebola virus disease and zika virus disease. (29, 30) . the secretary of the us department of health and human services declared a potential public health emergency on may 29, 2013, regarding mers-cov infection that could have a high potential to affect national security or the health and security of us citizens living abroad. the us food and drug administration (fda) subsequently issued an emergency use authorization to the centers for diseases control and prevention (cdc) for an in vitro molecular diagnostic test to diagnose mers-cov infection in multiple types of clinical specimens from symptomatic patients. the use of this test was later expanded to include the ability to test asymptomatic contacts of a person infected with mers-cov who traveled from saudi arabia to the united states. the cdc made this test available to multiple us public health laboratories, the us department of defense, and who laboratories worldwide. although the test has been distributed extensively, it is limited in terms of the cdc's ability to scale up the supply of reagents to support a surge in mers-cov cases in the united states and in other countries where the test has been made available. therefore, an emergency use authorization was issued on july 17, 2015, for the commercially developed realstar mers-cov rt-pcr kit u.s. (altona diagnostics gmbh, hamburg, germany) for use in the in vitro qualitative detection of mers-cov rna in tracheal aspirate or tracheal secretion samples (31) . although this commercial assay is a first step in bridging the diagnostic test availability gap in case of a surge scenario, the current coverage, at least in the united states, is insufficient until alternative, fda-cleared commercial tests are available (table 2) . a worldwide gap exists in the lack of readily available, simple, rapid, and accurate diagnostic tests for use in outpatient and inpatient clinical settings where the ability of the facility to use currently available, higher complexity molecular tests is limited. the lack of commercial development of mers-cov assays may be partially related to the limited availability of clinical specimens and mers-cov isolates from infected patients. availability of serum specimens from rt-pcrconfirmed mers-cov patients who survived can help facilitate development of serologic tests. if paired acute and convalescent serum samples are available, serologic tests can be used to confirm mers-cov infection when viral shedding is not detectable, and for surveillance purposes such as measuring population exposures and immunity to mers-cov infection. no investigational therapeutic drugs have been evaluated for treatment of mers-cov patients in prospective randomized controlled clinical trials. potential therapeutic drugs for mers-cov patients include available approved drugs with nonspecific properties, such as immunomodulators, small-molecule drugs with broad antiviral activity, repurposed fda-approved small-molecule drugs that show activity against mers-cov in vitro (table 3 ) (34, 35) , and newly developed monoclonal or polyclonal antibody therapies with specific activity against mers-cov (table 4 ) (54). one promising approach has been to investigate libraries of drugs approved by the fda and the european medicines agency. considering development times and manufacturing requirements for new products, repurposing of existing drugs might potentially facilitate a rapid response to outbreaks of emerging viruses (see regulatory section for a discussion on repurposing). other early-stage work on mers-cov therapeutics includes studies focusing on the essential viral replication steps of fusion, proteolysis, and rna polymerization (table 3 ) (54) . immunotherapeutics under evaluation consist of convalescent plasma and monoclonal and polyclonal antibodies. most of the monoclonal antibodies in development have specific neutralizing activity against the mers-cov spike protein (55, 56) . platforms are being developed to rapidly discover monoclonal antibodies, either from fully human convalescent blood or from transgenic animals, which can be manufactured on a large scale and are likely to have a good safety profile. the most advanced immunotherapeutic for mers-cov uses a transchromosomal bovine production system to produce fully human polyclonal mers-cov antibodies; a phase i study of this product was recently implemented (57; https://clinicaltrials.gov/ct2/ show/nct02788188). preliminary results from immunoprophylaxis or treatment studies have shown efficacy of fully human monoclonal or polyclonal antibodies in mers-cov-infected mice and nhps ( profile and a defined set of preclinical toxicology studies, challenges to development of immunotherapeutics include ensuring the absence of antibody-dependent enhancement of disease and reducing the risk for generation of escape mutant viruses that would be resistant to treatment. development of mers-cov candidate vaccines was initiated by the national institute for allergy and infectious diseases at the national institutes of health, academic investigators, and several companies (table 5 ). most candidate vaccines are still being evaluated in animal models. they have generally targeted the spike protein of mers-cov and are recombinant virus, subunit, dna, or virus-like vector vaccines (60,63-67). one live-attenuated mers-cov candidate vaccine is in early development (66) . preliminary studies for several other mers-cov vaccine candidates have been initiated, and early results demonstrate immunogenicity; 2 have progressed to nhp challenge, and a phase 1 clinical study in adults of 3 different doses of a dna plasmid vaccine that expresses the mers-cov spike protein was started in january 2016 (61) . ongoing assessment of antigenic evolution of circulating mers-cov strains is essential for informing vaccine development (68) . a concern that must be addressed in the development of mers-cov vaccines is the potential for causing antibody-dependent enhancement of disease upon virus challenge, such as what was observed with a sars-cov candidate vaccine upon sars-cov challenge (69) . the lack of a precedent of coronavirus vaccines for humans poses another challenge for the evaluation of mers-cov vaccines for humans, although vaccines against other animal coronaviruses are safe and in use in animals. considering the cultural importance of dromedaries on the arabian peninsula for meat, milk, and racing, prevention of camel-to-camel mers-cov transmission and reduction of spread from dromedaries to humans by camel vaccination is being investigated by government, academic, and commercial investigators (table 6 ). young camels appear to be at high risk for mers-cov infection and could be a priority group for vaccination (73, 74) ; the loss of maternal mers-cov antibodies ≈5-6 months after birth suggests a short time window for vaccination (75) . a major challenge to this approach is that dromedaries can be reinfected with mers-cov; a study by farag et al. found no correlation between mers-cov rna levels and neutralizing antibodies in camels (76) , suggesting that antibodies may not be protective against infection. because older camels can be reinfected, a camel vaccination strategy may require multiple dosing and booster vaccination to increase effectivein the united states and overseas (19) . in addition, 3 doses of a dna vaccine containing the mers-cov spike protein induced humoral immunity in dromedaries (60) . in a recent study, a modified vaccinia virus ankara vaccine that expresses the mers-cov spike protein was administered intranasally and intramuscularly to dromedaries; when challenged intranasally with mers-cov, vaccinated dromedaries had fewer signs of respiratory infection and lower mers-cov titers in the upper respiratory tract compared with unvaccinated dromedaries (77) . alpacas (new world camelids) are being investigated as a suitable proxy for camels because of the lack of available dromedaries in the united states, the high cost of acquiring dromedaries, and the relatively smaller size of alpacas (26, 27) . regulatory considerations for mers-cov medical countermeasures in the united states are focused on a pathway to human clinical trials for drugs and vaccines through submission of investigational new drug applications. investigational new drug submissions must adhere to requirements set forth in the code of federal regulations, title 21, part 312 (21 cfr 312; http://www.ecfr.gov/cgibin/text-idx?tpl=/ecfrbrowse/title21/21cfr312_main_02. tpl). several guidance documents exist on the fda website related to virology, microbiology, pharmacology and toxicology, and clinical and medical considerations (78). the most appropriate approval pathway is likely to be product-specific and will require consideration of existing product data, proposed intended use and population for use, and validated endpoints for efficacy predictive of clinical benefit, if any. likewise, data needed for consideration of an emergency use authorization, including dose finding and dose ranging, duration, and safety, can be obtained through sources such as investigational new drug clinical trials. repurposing of drugs approved by the fda for other illnesses for a mers-cov indication can potentially be expedited or accelerated if 1) the mechanism of action for antiviral activity is defined, 2) there is no change to the approved final drug form and route of administration, 3) dosing does not exceed the currently approved dose and duration for the currently indicated population and adequate pharmacokinetics data support this dosing, and 4) the risk-benefit profile is acceptable for the intended population and indication. for example, the risk-benefit profile for an approved drug with an oncology indication may be unacceptable if the drug is repurposed for administration to a healthy population for mers-cov postexposure prophylaxis. however, data requirements to initiate human trials will depend on the characteristics of the drug product and its intended use against mers-cov. as such, sponsors should consider prioritizing drug development on the basis of the totality of scientific evidence and merit of the drug alone, not on whether the drug has been previously approved. in the absence of a standardized and accepted animal model that simulates human disease from mers-cov infection, it is unclear how the fda may be able to expedite licensure or approval when data are lacking. the best approach may be collection of preclinical safety data and implementation of adaptive human clinical trials. this approach was taken for medical countermeasures in response to the 2013-2016 ebola virus disease outbreak. for diagnostic devices, the current emergency use authorization pathway serves as a fast approach to make products available for emergency public health purposes. after an emergency has been terminated, premarket notifications for these products should be submitted to fda for a more thorough evaluation as 510(k)s (http://www.fda.gov/ e6 emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 7, july 2016 medicaldevices/productsandmedicalprocedures/deviceap provalsandclearances/510kclearances/default.htm). the overarching goal for clinical research of mers-cov patients is to optimize clinical management and to identify effective therapies to improve survival. although clinical data on some mers-cov patients have been published in case series (58, 79, 80) , there is a need for much more epidemiologic, clinical, virologic, and immunologic data to improve the limited understanding of the pathogenesis of mers-cov infection in humans. gaps include information on viral load and duration of viral shedding in blood, urine, respiratory, and other clinical specimens from infected persons; understanding of the innate and adaptive immune response to mers-cov infection; pathology data on the distribution of mers-cov in respiratory and extrapulmonary tissues in fatal cases; information from autopsies of persons who died of mers-cov; and an overall improved understanding of the pathogenesis of mers-cov in humans. only 1 study has investigated mers-cov infection in autopsy tissues of a patient who died from the disease (81) . collaborations are especially needed to pool and systematically collect serial clinical specimens from mers-cov patients for virologic, immunologic, and biomarker analyses to correlate with clinical illness, and to conduct long-term follow-up of survivors of severe disease (82) (83) (84) . detailed understanding of host factors and cofactors associated with disease severity from asymptomatic infection to fatal illness is needed. efforts to promote international sharing of clinical specimens and mers-cov isolates are needed to foster development of diagnostics, therapeutics, and vaccines. use of standardized clinical data collection instruments and common biologic sampling protocols for serial prospective data collection will facilitate data pooling from mers-cov cases and comparisons across clinical sites and countries. global collaborations among clinical networks are also needed to implement clinical trials, preferably randomized controlled clinical trials, of mers-cov investigational therapeutics (82) (83) (84) (85) . without an international agreement on protocols and systematic standardization of case reporting and data collection methods, haphazard or anecdotal reporting and analysis of disease course and outcome may continue. who and the international severe acute respiratory and emerging infection consortium are collaborating in adapting standardized protocols for controlled clinical trials for mers-cov (83) . prospective controlled clinical trials (ideally randomized clinical trials) of potential mers-cov therapies and vaccines in humans are needed urgently; however, there is uncertainty in estimating timelines for the development of potential mers-cov medical countermeasures because of the need to further characterize existing and new animal models, the unpredictability of demonstrating a favorable risk-benefit outcome during preclinical testing, and competition for resources with other emerging infectious diseases. in addition, the risk for antibody-dependent enhancement of disease may interrupt the timeline for conducting human clinical trials of mers cov vaccines and immunotherapeutics. researchers of all potential mers-cov medical countermeasures should have preclinical toxicology data available before initiating human clinical trials. although animal efficacy data are not technically required before implementing human clinical trials of potential countermeasures, such data are considered important for identifying the most promising medical countermeasure candidates, justifying risk in human volunteers, and informing the design of future clinical studies. timeframes for the production of specimen panels and repositories to aid commercial diagnostic development are also contingent on obtaining adequate funding and clinical samples. although preclinical development and research on potential mers-cov medical countermeasures has achieved appreciable progress to date, such development is preliminary, and substantive challenges must be overcome before most potential countermeasures are ready for human emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 7, july 2016 e7 results of substantial progress in establishing the infrastructure and platforms for preclinical and advanced clinical development of countermeasures can serve as a model to enable more timely response to other emerging infectious diseases of global public health concern in the future. world health organization. emergencies preparedness, response: middle east respiratory syndrome coronavirus saudi arabia world health organization. summary of current situation, literature update and risk assessment hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus (mers-cov)-republic of korea world health organization. middle east respiratory syndrome coronavirus (mers-cov)-china rapid medical countermeasure response to infectious diseases: enabling sustainable capabilities through ongoing public and private-sector partnerships rapid generation of a mouse model for middle east respiratory syndrome generation of transgenic mouse model of middle east respiratory syndrome-coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels animal models for sars and mers coronaviruses a comparative review of animal models of middle east respiratory syndrome coronavirus infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection animal models of middle east respiratory syndrome coronavirus infection treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a non-human primate model of common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas. emerg infect dis experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus. emerg infect dis mers-cov infection of alpaca in a region where mers-cov is endemic viral shedding and environmental cleaning in middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications regulatory and policy framework-emergency use authorization; middle east respiratory syndrome coronavirus (mers-vov) eus information assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections first international external quality assessment of molecular diagnostics for mers-cov screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study public summary of opinion on orphan designation. interferon alfa-n3 for the treatment of middle east respiratory syndrome systematic review of treatment effects protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 evaluation of ssya10-001 as a replication inhibitor of sars, mhv and mers coronaviruses anti-infective immunoadhesins from plants protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors anti-mers-cov convalescent plasma therapy cows with human chromosomes enlisted to fight hantavirus exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution a conformationdependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in mers-cov spike protein inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus coronaviruses-drug discovery and therapeutic options 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection evaluation of candidate vaccine approaches for mers-cov a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates phase i, open label dose ranging safety study of gls-5300 in healthy volunteers middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus camels emit dangerous mers virus, csu confirms mers surges again, but pandemic jitters ease immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice mers-cov in upper respiratory tract and lungs of dromedary camels mers coronavirus in dromedary camel herd, saudi arabia time course of mers-cov infection and immunity in dromedary camels high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in united arab emirates middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways international severe acute respiratory and emerging infection consortium. severe acute respiratory infection data tools open source clinical science for emerging infections clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance we thank john beigel, wendy carter, david cho, su-young choi, eric donaldson, heinz feldmann, barney graham, lisa hensley, cynthia kelley, peter miele, vincent munster, david spiro, kanta subbarao, and melissa willis for participating in foundational discussions and for help assessing potential therapeutic drugs and methods; vaccines under development or investigation; timelines for animal studies; scale-up of medical countermeasures; when human trials are to be started; regulatory issues for investigational new drug (ind) and emergency use authorization (eua) applications, and clinical studies; and safety issues and prioritization for clinical trials. we also thank dean erdman for insightful discussions on mers-cov diagnostic assay development, rick bright, tom dreier, and byron rippke for helpful commentary, and thuy phan for organizational assistance.dr. uyeki is the chief medical officer for the influenza division, national center for immunization and respiratory diseases, cdc. his research interests are in the epidemiology and clinical management of influenza and emerging infectious diseases. key: cord-332237-8oykgp0h authors: omrani, ali s; saad, mustafa m; baig, kamran; bahloul, abdelkarim; abdul-matin, mohammed; alaidaroos, amal y; almakhlafi, ghaleb a; albarrak, mohammed m; memish, ziad a; albarrak, ali m title: ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study date: 2014-09-29 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70920-x sha: doc_id: 332237 cord_uid: 8oykgp0h background: middle east respiratory syndrome coronavirus (mers-cov) infection is associated with high mortality and has no approved antiviral therapy. we aimed to compare ribavirin and interferon alfa-2a treatment for patients with severe mers-cov infection with a supportive therapy only. methods: in this retrospective cohort study, we included adults (aged ≥16 years) with laboratory-confirmed mers-cov infection and pneumonia needing ventilation support, diagnosed between oct 23, 2012, and may 1, 2014, at the prince sultan military medical city (riyadh, saudi arabia). all patients received appropriate supportive care and regular clinical and laboratory monitoring, but patients diagnosed after sept 16, 2013, were also given oral ribavirin (dose based on calculated creatinine clearance, for 8–10 days) and subcutaneous pegylated interferon alfa-2a (180 μg per week for 2 weeks). the primary endpoint was 14-day and 28-day survival from the date of mers-cov infection diagnosis. we used χ(2) and fischer's exact test to analyse categorical variables and the t test to analyse continuous variables. findings: we analysed 20 patients who received ribavirin and interferon (treatment group; initiated a median of 3 days [range 0–8] after diagnosis) and 24 who did not (comparator group). baseline clinical and laboratory characteristics were similar between groups, apart from baseline absolute neutrophil count, which was significantly lower in the comparator group (5·88 × 10(9)/l [sd 3·95] vs 9·88 × 10(9)/l [6·63]; p=0·023). 14 (70%) of 20 patients in the treatment group had survived after 14 days, compared with seven (29%) of 24 in the comparator group (p=0·004). after 28 days, six (30%) of 20 and four (17%) of 24, respectively, had survived (p=0·054). adverse effects were similar between groups, apart from reduction in haemoglobin, which was significantly greater in the treatment group than in the comparator group (4·32 g/l [sd 2·47] vs 2·14 g/l [1·90]; p=0·002). interpretation: in patients with severe mers-cov infection, ribavirin and interferon alfa-2a therapy is associated with significantly improved survival at 14 days, but not at 28 days. further assessment in appropriately designed randomised trials is recommended. funding: none. since it was fi rst described in september, 2012, 855 cases of middle east respiratory syndrome coronavirus (mers-cov) infection have been confi rmed, 333 of which were fatal. 1,2 cases occur sporadically, as community clusters or as hospital outbreaks, and range in severity from asymptomatic or mild illness to rapidly progressive and fatal disease. [2] [3] [4] [5] [6] the management of patients with mers-cov infection consists of a combination of supportive measures, antimicrobial therapy for any associated bacterial or viral infections, and strict implementation of appropriate infection control precautions. 7 so far, no antiviral therapy has been approved for the treatment of patients with mers-cov infection. 8 several therapeutic interventions for coronavirus were investigated during the large multinational outbreak of severe acute respiratory syndrome (sars) in 2003. 9, 10 reviews of the available scientifi c literature suggest that a combination of ribavirin and interferon might be of benefi t in patients with severe mers-cov infection. 8, 11, 12 furthermore, this combination was shown to inhibit mers-cov in cell culture and seemed to improve outcomes in an animal study. 13, 14 both agents are associated with substantial potential adverse eff ects and hence their clinical use should be carefully balanced against any potential harm. 8 we aimed to assess outcomes of a treatment programme for patients with severe mers-cov infection that consisted of oral ribavirin and subcutaneous pegylated interferon alfa-2a. we report the results and outcomes in patients given treatment in accordance with this protocol by comparison with a historical group who received supportive therapy only. this single-centre, retrospective cohort study included individuals who were diagnosed with laboratoryconfi rmed mers-cov infection between oct 23, 2012, and may 1, 2014, at the prince sultan military medical city (riyadh, saudi arabia). eligible patients were those aged 16 years or older with severe pneumonia needing invasive or non-invasive ventilation. no exclusion criteria were applied at this stage. mers-cov infection was diagnosed by rt-pcr testing of respiratory tract samples for mers-cov upe, orf 1b, and n genes. 15 all rt-pcr tests for mers-cov were done at the saudi ministry of health regional laboratory in jeddah and riyadh, saudi arabia. pneumonia was defi ned as new, otherwise unexplained, lower respiratory tract symptoms such as cough or shortness of breath with at least one systemic feature such as fever or chills, and new focal chest signs on examination, in addition to new or progressive pulmonary infi ltrates on chest radiograph. 16 from sept 16, 2013, all eligible patients were off ered treatment with oral ribavirin and subcutaneous pegylated interferon alfa-2a after informed written consent had been obtained from the patients themselves or their next of kin. the treatment protocol was approved by pharmacy and therapeutics committee. the study was approved by the research ethics committee at the prince sultan military medical city to allow retrospective access to patients' records and fi les. pegylated interferon alfa-2a (pegasys; roche pharmaceuticals, basel, switzerland) was given by subcutaneous injection at a dose of 180 μg per week for 2 weeks. the dose of oral ribavirin (copegus; roche pharmaceuticals) was adjusted according to calculated creatinine clearance and continued for 8-10 days. 12 patients with a creatinine clearance of greater than 0·833 ml/sec/m 2 received a 2000 mg loading dose, followed by 1200 mg every 8 h for 4 days then 600 mg every 8 h for 4-6 days; those with a creatinine clearance of 0·333-0·833 ml/sec/m² received a 2000 mg loading dose, followed by 600 mg every 8 h for 4 days then 200 mg every 6 h for 4-6 days; and those with a creatinine clearance of <0·333 ml/sec/m² or on dialysis received a 2000 mg loading dose, followed by 200 mg every 6 h for 4 days then 200 mg every 12 h for 4-6 days. patients did not receive ribavirin and interferon alfa-2a therapy if they were diagnosed before sept 16, 2013, or if they declined consent. all patients received appropriate supportive care such as supplementary oxygen, vasopressor therapy, and renal replacement as needed. hydrocortisone 200 mg daily was given to patients with refractory septic shock and continued until vasopressor therapy was no longer needed. 17 in addition to regular clinical monitoring, renal function, liver enzymes, and blood count were assessed at baseline and daily throughout the treatment course. conscious patients were monitored for any clinical signs of depression or acute confusion. patients who received ribavirin and interferon alfa-2a therapy were classifi ed as being in the treatment group and those who did not made up the comparator group. two investigators, aso and kb, both of whom were masked to group allocation and the patients' clinical outcomes, compared baseline characteristics of the two groups. immunosuppressive therapy 1 (5%) 4 ‡ (18%) 0·66 data are number (%) or mean (sd). apache ii=acute physiology and chronic health evaluation ii. sofa=sequential organ failure assessment. *only 19 patients were assessed. †only 13 patients were assessed. ‡only 22 patients were assessed. the primary endpoints for the study were 14-day and 28-day survival from the date of mers-cov infection was diagnosis. we used χ² and fischer's exact tests for categorical variables, whereas we used the student's t test for continuous variables to assess the diff erences in means of the two groups. the log-rank test was used for assessing survival diff erences between the two groups. our cutoff for statistical signifi cance was 0·05. the graphical and statistical tests suggested that the pro portionalhazard assumption was not violated. we did statistical analyses using microsoft excel 2011 and stata statistical software, release 12. no external funding was received for this study. zm had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. 70 individuals were diagnosed with mers-cov infection between oct 23, 2012, and may 1, 2014. baseline characteristics were generally similar between patients who received ribavirin and interferon alfa-2a therapy and those who did not (table 1) , with the exception that end-stage renal failure was present in three patients who did not receive study treatment and in none who did. after excluding ineligible patients, 44 patients were included in the study: 20 in the treatment group and 24 in the comparator group (fi gure 1). the mean age of all 44 patients was 65·5 years (sd 18·2), and 32 (73%) were men (table 1) mean absolute neutrophil count was signifi cantly lower in the treatment group than in the comparator group (table 1) ; however, no other statistically signifi cant differences in baseline characteristics or support measures were noted between the two groups (tables 1, 2). of all 44 patients, 21 (48%) were still alive 14 days after diagnosis of mers-cov infection, whereas at 28 days only ten (23%) had survived. 14 (70%) of 20 patients in the treatment group were alive 14 days after diagnosis, compared with seven (29%) of 24 in the comparator group (p=0·004). however, six (30%) of 20 patients in the treatment group survived up to 28 days from diagnosis of mers-cov infection, whereas four (17%) of 24 did in the comparator group (p=0·054; fi gure 2). ribavirin and pegylated interferon therapy was well tolerated by the treatment group with no premature discontinuation secondary to adverse eff ects. however, the mean drop in haemoglobin over the treatment course was signifi cantly greater in the treatment group (4·32 g/l [sd 2·47]) than in the comparator group eff ective treatment interventions for patients with severe mers-cov infection are still urgently needed. in critically ill patients with severe mers-cov infection, our study shows that ribavirin and pegylated interferon alfa-2a therapy is associated with a signifi cant 14-day survival benefi t compared with standard treatment. 28-day survival also seemed to improve with ribavirin and pegylated interferon alfa-2a therapy, but the diff erence between groups was not signifi cant (panel). the loss of a signifi cant survival diff erence over time might be partly explained by most patients in our cohort having several comorbidities with high apache ii and sofa scores. mortality is known to be very high in patients with severe mers-cov infection who need critical-care support. 24 therefore, long-term survival benefi t, if present, might be diffi cult to show in smaller studies. treatment with ribavirin and interferon was well tolerated in our study. the only adverse event that was signifi cantly worse in the treatment group was mean decrease in haemoglobin (4.32 g/l in the treatment group compared with 2·14 g/l in the comparator group). anaemia is a well recognised complication of ribavirin therapy and was noted previously in studies investigating the role of ribavirin in the treatment of sars coronavirus infection. 25, 26 of note, receipt of packed red blood cells was not signifi cantly diff erent between the treatment and comparator groups in our study. furthermore, no treatment discontinuations occurred as a result of anaemia. therefore, the risk of ribavirin-associated anaemia-although substantial and in need of careful monitoring-might not hinder the use of ribavirin for patients with severe mers-cov infection, especially if a survival benefi t can be confi rmed. baseline absolute neutrophil count was signifi cantly lower in the treatment group and therefore a signifi cantly lower minimal absolute neutrophil count during the course of the illness is not surprising. several investigators showed that interferon α has useful in-vitro activity against mers-cov. 13, 20, 26 however, when compared with interferon α and interferon γ, interferon β seems to have the most potent inhibitory in-vitro activity against mers-cov. 21 ribavirin has slight anti-mers-cov activity in vitro when used alone or in combination with interferon α. 13, 22 mycophenolic acid is another compound that exhibits signifi cant in-vitro activity against mers-cov. 21 of 290 compounds screened, 60 were active in cell culture against mers-cov. 23 although only the combination of interferon α plus ribavirin has so far undergone in-vivo assess ment against mers-cov, 14 many others are potential candidates for further clinical assessment. one of the limitations of our study is its small size. however, only two previous reports of clinical use of ribavirin and interferon for mers-cov infection have been published. 18, 19 in a retrospective report, fi ve patients with severe mers-cov infection, all of whom had signifi cant comorbidities and needed mechanical ventilation, received a com bination of ribavirin and pegylated interferon alfa-2b a median of 19 days after admission. none of the patients survived and the investigators concluded that late commencement of therapy might not be benefi cial. 18 in another report, 19 a patient with severe mers-cov infection received ribavirin and interferon therapy with good clinical response and no signifi cant adverse eff ects. our study, albeit small, is the largest clinical investigation so far to assess the use of this combination in the treatment of patients with severe mers-cov infection. although baseline characteristics of our treatment and comparator groups seem to be reasonably balanced, substantial diff erences might not be apparent because of the small number of patients in the study. our study is also limited by its retrospective, nonrandomised nature. inevitably, selection and unmeasured confounding bias cannot be completely excluded. undoubtedly, new interventions should ideally be assessed in randomised, controlled clinical trials. however, such an approach is generally accepted to not always be practically feasible in the context of an emerging and relatively uncommon disease such as mers-cov infection. 8 we carefully selected our comparator group, ensuring that the two cohorts were matched as closely as possible in their clinical characteristics and treatment interventions other than the receipt of ribavirin and interferon. we removed three individuals who had outlying baseline serum creatinine from the comparator group to minimise the risk of any spurious conclusions driven by clinical characteristics that might be potentially detrimental to clinical outcome. clinical outcomes for each individual were masked from investigators who selected patients and did matching assessments. the absence of serial viral load measurement in lower respiratory tract samples in our study makes it impossible to show any association between temporal viral load changes and antiviral therapy. such measurements should be included in any future clinical studies exploring the therapeutic benefi t of any antiviral intervention for patients with mers-cov infection. severe mers-cov is associated with poor overall survival. treatment with oral ribavirin and subcutaneous pegylated interferon alfa-2a is associated with signifi cantly improved survival at 14 days, but not at 28 days. the combination is associated with signifi cant falls in haemoglobin, but no other signifi cant adverse eff ects were noted. treatment with ribavirin and pegylated interferon might be considered in patients with severe mers-cov infection, provided that adequate monitoring and assessment can be ensured. further assessment, including in patients with less severe mers-cov infection, in appropriately designed randomised trials, is recommended. this study was initiated and designed by aso, mms, zam, and ama. aso, mms, ab, ma-m, aya, gaa, mma, zam, and ama obtained and collated patient data. kb undertook all statistical analyses for the study. aso and kb prepared all tables and fi gures. aso, mms, zam, and ama wrote the fi rst draft of the manuscript and all authors reviewed and contributed to subsequent drafts and the fi nal report. aso has received consultancy fees from gilead, pfi zer, msd, and viiv; payment for lectures from pfi zer, msd, glaxosmithkline, and sanofi -aventis; and sponsorship to attend international meetings and conferences from msd, pfi zer, biopharma, bristol-myers squibb, and janssen-cilag. ab has received travel funding to attend an international meeting from pfi zer. gaa has received travel funding to attend an international meeting from edwards lifesciences. all other authors declare no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov)-11th update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case hospital outbreak of middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical 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middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon (ifn)-alpha-2b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada severe acute respiratory syndrome: report of treatment and outcome after a major outbreak we thank staff of prince sultan military medical city, riyadh, saudi arabia, for the clinical care given to the patients and for facilitating access to the relevant medical records. we searched pubmed for reports published in english any time before june 24, 2014, with the search term "[(mers-cov or hcov-emc or novel coronavirus) and (therapy or interferon or ribavirin]". we found one animal study, 14 two small case series in human beings, 18, 19 and several in-vitro studies. 13, [20] [21] [22] [23] the data suggested that combination therapy with ribavirin and interferon alfa could have potential benefi ts for patients with severe mers-cov infection. this is, to our knowledge, the largest clinical study done so far assessing the potential benefi t and safety of combination therapy with pegylated interferon alfa-2a plus ribavirin in patients with severe mers-cov infection. because we noted a signifi cant 14-day survival benefi t in patients who received the combination compared with those who received supportive therapy only, but no survival benefi t at 28 days, we recommend further assessment in appropriately designed randomised clinical trials to provide further information about the role of this combination in the treatment of patients with severe mers-cov infection. key: cord-316194-jnw8gr7e authors: sifuentes, monica title: chapter 61 disorders of the ear date: 2005-12-31 journal: pediatrics doi: 10.1016/b978-0-323-01199-0.50066-9 sha: doc_id: 316194 cord_uid: jnw8gr7e nan disorders of the ear in pediatric patients include infections of the middle ear, infections of the external ear canal, congenital and acquired masses, and complications related to blunt or direct head trauma. early and correct recognition of these conditions is essential. otitis media (om) is an acute or chronic inflammation of the middle ear. acute om (aom) is an acute infection of the middle ear with middle ear effusion (mee) by otoscopic examination. the tympanic membrane (tm) often is red, bulging, and opaque. aom usually is manifested by ear pain or tugging or rubbing of the ear. fever and irritability may be present. om with effusion (ome) is a painless collection of fluid in the middle ear without any signs of acute inflammation or infection. most cases evolve from aom, but a preceding infection is not necessary. recurrent aom is defined as three new episodes of aom within 6 months or four episodes within 1 year. resolution of the infection should be documented between each episode of aom, but this may be difficult if ome develops. chronic om is the presence of mee for more than 3 months. recurrent and chronic disease can occur simultaneously because the presence of one condition often predisposes to the other. complications of om can be divided into intratemporal and intracranial causes (table 61 -1). children differ from adults in that complications are more likely to occur from aom than chronic ear disease and often are the result of delayed treatment. hearing loss, the most prevalent complication, can be conductive, sensorineural, or mixed. the magnitude of the conductive hearing loss ranges from 15 to 40 db and seems to result from the quantity, rather than the quality, of fluid in the middle ear. this type of hearing loss disappears when the mee resolves. infectious or inflammatory mediators in the middle ear or in the labyrinth can cause sensorineural hearing loss. children with aom usually have fluid present in the mastoid air cells because of the direct connection with the middle ear ( fig. 61-1) . mastoiditis, an inflammatory process that accompanies om, develops when there is destruction of bone or formation of a subperiosteal abscess within the cavity. early in its course, mastoiditis may resolve spontaneously. when the infection persists for more than 1 week, inflammatory granulation tissue forms, and a series of changes occurs within the cavity, resulting in either acute or chronic mastoiditis. acute mastoiditis is subdivided into pathologic stages depending on the progression of the disease within the mastoid air cells. chronic mastoiditis almost always is associated with chronic suppurative om, a chronic (≥2 months) drainage from the middle ear and the mastoid through either a perforation of the tm or a tympanostomy tube. the most common suppurative intracranial complication of om and mastoiditis is bacterial meningitis, although its incidence has decreased dramatically since the advent of antibiotic therapy for acute infections and routine immunization against haemophilus influenzae type b. other 6 consult otolaryngologist as needed for assistance with medical and surgical management. 7 prevent long-term sequelae associated with untreated disorders of the ear, including sensorineural hearing loss. 8 provide appropriate follow-up for children who have recurrent otitis media, hearing loss, and traumatic perforations of the tympanic membrane. intracranial complications, such as subdural empyema and brain abscess, occur rarely in developed countries and are associated more often with other chronic diseases, such as sinusitis, rather than aom. otitis externa (oe) refers to inflammation and infection of the external ear canal or the auricle. it is commonly known as swimmer's ear, an acute diffuse inflammation of the external auditory epithelium extending from the pinna all the way to the tm. although inflammation of the tm also may occur, it usually can be differentiated from aom by pneumatic otoscopy. perforations of the tm may be temporary or permanent depending on whether they are associated with aom, a chronic infection, a surgical procedure, or trauma. permanent perforations are classified as central or marginal. in central perforations, the fibrous annulus is unaffected, and the tm circumscribes the entire intact ring. marginal perforations involve the fibrous annulus, and the defect is seen posteriorly on the tm. abnormal growth of squamous epithelium into the middle ear as a result of destruction of the margin of the tm may lead to formation of a secondary cholesteatoma. the most common ear mass in children is a cholesteatoma, a histologically benign lesion in the middle ear, mastoid space, or petrous bone containing keratinizing squamous epithelium. occasionally a cholesteatoma may be found in the external auditory canal. congenital, or primary, cholesteatoma is defined as the presence of squamous epithelium medial to an intact tm without a significant past history of aom or eustachian tube dysfunction. acquired, or secondary, cholesteatoma occurs more commonly as a complication of chronic om. cholesteatomas are locally invasive and can destroy important structures, such as the ossicles, cochlea, or semicircular canals. figure 61 -1 illustrates the normal anatomy of the ear and the relationship of the external, middle, and inner ear to one another. the location of the eustachian tube acts as a conduit connecting the middle ear to the nasopharynx. the development of om in children is the direct result of abnormal function of this tube. anatomic features, such as its length and position relative to the posterior nasopharyngeal wall, contribute to increased frequency of om. the eustachian tube in young infants is shorter and more horizontal than in older children ( fig. 61-2) . the eustachian tube has three major physiologic functions: (1) ventilation of the middle ear, (2) drainage of middle ear fluid, and (3) protection of the middle ear from nasopharyngeal secretions. these functions occur via active dilation of the tube by contraction of the tensor veli palatini muscle ( fig. 61-3 ). normal function of this muscle is essential in preventing mee and acute infections. eustachian tube dysfunction is the result of abnormal patulence of the tube, mechanical obstruction ( fig. 61-4) , or both. functional obstruction results from poor tensor veli palatini function. mechanical obstruction occurs from either an extrinsic or an intrinsic cause. extrinsic causes include enlarged adenoids or the presence of a nasopharygeal tumor. intrinsic causes often are related to injury or inflammation of distal tubal epithelium by upper respiratory infections with viruses such as influenza or respiratory syncytial virus. epithelial damage leads to increased mucus secretion and cellular debris, which then obstructs the lumen. if the eustachian tube is occluded, negative middle ear pressure develops as oxygen is absorbed from this space. transudative serous capillary fluid accumulates in the middle ear and the mastoid air cells and can be infected with nasopharyngeal bacteria that enter through the eustachian tube as it opens intermittently. replication of the bacteria within the serous fluid leads to a series of inflammatory processes with the release of bacterial and host cell products into the middle ear. the clinical manifestation of these events is aom. some infants and young children are at increased risk for developing om. well-documented environmental risk factors include passive tobacco smoke exposure, lack of breast-feeding, bottle-propping, poor socioeconomic status, and day care attendance. children with craniofacial anomalies or congenital or acquired immunodeficiency syndromes have a higher incidence of om and recurrent disease, as do certain ethnic groups, including whites, alaskan eskimos, and native americans. the peak incidence of aom occurs during the first 2 years of life, particularly between 6 and 12 months of age. another peak occurs after the time of school entry. in temperate areas of the world, most infections occur during the fall, winter, and early spring, seasons when most viral upper respiratory infections occur in young children. recurrent disease after age 5 years should prompt a more extensive evaluation to exclude other predisposing factors, such as allergies or an underlying immunologic condition. streptococcus pneumoniae, nontypable h. influenzae, and moraxella catarrhalis account for approximately 85% of acute infections. gram-negative enteric bacilli account for about one fifth of middle ear effusions in infants younger than 6 weeks old. other, less common bacteria reported to cause aom include group a, c, and g streptococci; staphylococcus aureus; streptococcus epidermidis; and mixed flora. the role of viral pathogens has become much more apparent with more sensitive laboratory detection methods. common virus isolates from middle ear fluid in acute infections include respiratory syncytial virus, influenza a and b, rhinovirus, adenovirus, coronavirus, and parainfluenza (types 1, 2, and 3). some studies report that 25% of middle ear effusions are "sterile." approximately 70% of isolates from chronic mee yield bacterial growth. in mee pseudomonas aeruginosa and coagulase-negative staphylococci play a prominent role. mastoiditis is an extension of the acute inflammatory process of om. at birth, the mastoid air cell system comprises a single cell, the antrum, which is connected to the middle ear by a small channel called the aditus ad antrum. pneumatization of the mastoid bone begins soon after birth and continues throughout life. by age 2, this process is usually extensive. all mastoid air cells end up interconnected with the antrum, which sometimes is referred to as the bottleneck when an acute infection cannot drain from the mastoid into the middle ear. clinically significant disease develops when an infection within the mastoid cavity spreads to the periosteum covering the mastoid process. with further progression, the bony trabeculae that separate the mastoid air cells are destroyed, resulting in a mastoid empyema. invasion of adjacent structures leads to formation of a soft tissue, subperiosteal abscess, or chronic mastoiditis. rarely, further extension into the petrous portion of the temporal bone may occur, resulting in petrositis. a classic triad of retrobulbar pain, persistent otorrhea, and ipsilateral abducens nerve palsy (gradenigo's syndrome) is seen in patients with this complication. infection also can spread to the labyrinth and facial nerve or into the intracranial cavity causing more serious morbidity and mortality. acute mastoiditis occurs most commonly as a complication of chronic suppurative om but can develop after an acute infection. acute tm perforation and the presence of a cholesteatoma are predisposing factors. currently the incidence of mastoiditis is reportedly less than 0.1%. the incidence may change with recommendations to use oral antibiotics only in children with unequivocal acute infections of the middle ear. countries in which acute om is observed initially, rather than treated with antimicrobials, do not have a higher incidence of mastoiditis. most cases of acute mastoiditis are caused by the same organisms that cause aom. other pathogens include streptococcus pyogenes and s. aureus. in subacute or chronic cases, s. aureus and gram-negative enteric bacilli, such as escherichia coli, proteus, and p. aeruginosa, are common isolates from persistent and indolent infections. the external ear consists of the pinna and the external auditory canal, which consists of a lateral cartilaginous portion (approximately one third of the canal) and a medial osseous portion. before development of the osseous portion during infancy, the external auditory meatus is predominantly cartilaginous. the squamous epithelium is thicker over the lateral cartilaginous portion of the canal compared with the osseous portion from subcutaneous tissue containing hair follicles and sebaceous and ceruminous glands. the sebaceous glands, located superficially in the dermis, secrete an oily substance called sebum, whereas the apocrine (ceruminous) glands secrete a milky opaque fatty fluid. cerumen is a mixture of these two secretions and desquamated epithelial cells. its purpose is to form a protective acidic lipid layer that limits pathogenic bacterial overgrowth and inhibits maceration from water or sweat. oe develops from alterations in the ph of the external auditory canal, local trauma, or secondary maceration of the skin. high humidity, increased environmental temperature, and water contamination increase the ph of the canal and subsequent risk for acute oe. moisture in the canal raises the ph and removes the protective lipid layer of the skin, wb saunders, 1988.) leading to edema and maceration of the epithelial lining. excessive sweating, absence of cerumen, hearing aid irritation or improper fit, earplug usage, and the insertion of a foreign body to scratch the ear canal also can play a role in the development of infection. water that has high bacterial counts and the ph of pool water play an important role in oe rather than the number of bacteria in the water. hot tubs, whirlpools, and pressurized ear irrigation are other sources of water contamination. oe is seen most often in the summer months in temperate climates and year round in warmer areas. most cases are unilateral, with mild-to-moderate diffuse inflammation. rarely, in severe cases, infection may extend to the surrounding soft tissue and lymph nodes. the organisms primarily responsible for acute oe are p. aeruginosa and s. aureus. other organisms include s. epidermidis, proteus, enterobacter, klebsiella, and streptococci. many acute infections are polymicrobial, with aerobic and anaerobic bacteria. acute otomycosis (fungal oe) accounts for 10% of cases of oe and is caused most often by aspergillus niger. acute tympanic perforations are secondary to trauma or aom. certain racial groups may experience perforations with aom at a higher rate than the general population. spontaneous eardrum perforation has been reported with almost every episode of aom in eskimos and native americans. chronic perforations occur under a variety of circumstances, including (1) when an acute perforation fails to heal after an episode of aom, (2) after spontaneous extrusion (or removal) of a tympanostomy tube, (3) after long-standing atelectasis of the tm, and (4) when an acute traumatic perforation fails to close. small chronic perforations, regardless of their location, have little impact on hearing in the absence of other abnormalities. large perforations can be associated with a 20-to 30-db conductive hearing deficit. traumatic perforations of the tm occur from rapid changes in ambient pressure that occur with certain sports, such as diving, water-skiing, and surfing, or with activities such as flying in an unpressurized airplane. blunt trauma with child abuse (e.g., the slap of an open hand against a child's ear by an angry parent) can rupture the tm. tm lacerations may occur by the accidental or intentional placement of a foreign body, such as a cotton-tip applicator or hairpin, into the external auditory canal. some injuries are severe enough to damage the ossicular chain or cause a perilymphatic fluid leak or fistula. congenital cholesteatoma is an asymptomatic keratotic white mass located behind an intact membrane in a patient with no significant history of recurrent middle ear disease or previous ear surgery. although the origin of the lesion is not well understood, it is presumably present since birth. the most widely accepted theory is that congenital cholesteatoma results from a persistent epidermoid formation in the developing middle ear that, under normal circumstances, disappears after 33 weeks' gestation. the most common age of diagnosis is 4 years, but this also can range from infancy to adolescence. boys are affected more commonly than girls in a ratio of 3:1. most acquired cholesteatomas occur as a complication of chronic om and arise as a focal area of retraction of the tm. three main theories exist to explain their patho-genesis: (1) implantation and invasion of squamous epithelium into the middle ear secondary to ear surgery or temporal bone fracture, (2) migration and invasion of squamous epithelium through a perforation of or retraction of the tm, and (3) metaplasia of low cuboidal epithelium of middle ear mucosa caused by infection. regardless of the etiology, certain children are at increased risk for the development of secondary cholesteatoma, including children with cleft palate, trisomy 21, and aural atresia or stenosis. in the case of aural atresia, occult lesions can develop in the remnant of the external auditory canal or in the middle ear cleft. in the verbal child, the diagnosis of aom is usually straightforward, with the patient complaining of ear pain (otalgia), fever, and an antecedent upper respiratory infection. the affected tm appears markedly erythematous or injected and often is opaque and thickened, obscuring visualization of the bones (ossicles) in the middle ear. the tm is "full" or bulging. an air-fluid level also may be seen. preverbal children and infants are a greater diagnostic challenge. symptoms often are nonspecific, such as irritability, poor sleeping, and decreased appetite. unaccustomed ear tugging or rubbing can be seen, but is inconsistently predictive. the cartilaginous external ear canal of an infant can be tortuous, making visualization of the tm difficult. accurate assessment of the color and opacity of the tm is particularly hard in an apprehensive, febrile, crying toddler, sometimes requiring the use of additional diagnostic procedures, such as tympanometry. tympanometry is an objective test that measures the mobility, or compliance, of the tm and the middle ear. it does not evaluate hearing but is sensitive in detecting mee. compliance is measured via a probe tone presented to the sealed canal. compliance is reported as high (≥ 0.5 ml), intermediate (<0.5 ml but >0.2 ml), or low (≤ 0.2 ml). a tympanogram also measures middle ear pressure, which is categorized as normal, negative, or positive and represented as a curve, or peak, on a graph (reported in mm h 2 o). a peak between −100 mm h 2 o and +50 mm h 2 o is normal. a peak less than −100 mm h 2 o signifies high negative pressure, and a peak at greater than +50 mm h 2 o is consistent with high positive pressure. a sharp tympanometric peak suggests a low likelihood of mee; a rounded one suggests a greater likelihood of fluid. a flat one is highly suggestive of mee. tympanometry alone cannot distinguish between an acute infection of the middle ear and an uninflamed effusion (ome). the highly compliant cartilaginous walls of the external canal of infants younger than 6 months old can expand when air pressure is increased in the canal in this age group and result in a falsely normal reading. all examinations of the middle ear should include a determination of the mobility of the tm via pneumatic otoscopy. normal mobility is shown when positive pressure is applied in the external auditory canal and the tm moves rapidly inward (away from the examiner). when the bulb is released, negative pressure is created, and the tm moves outward (toward the examiner). mobility of the tm is reduced greatly by fluid or pus in the middle ear. tympanocentesis, needle aspiration of the middle air space, is a diagnostic and therapeutic procedure. it may be indicated to determine the causative organism of a middle ear effusion in infants younger than 6 weeks old, children who are immunocompromised, or children who are toxic or have signs of invasive bacterial disease. presence of suppurative complications also may warrant this procedure. tympanocentesis may be indicated if the diagnosis of aom is unclear or if the patient is not improving after appropriate therapy. potential complications include chronic perforation of the tm, facial nerve paralysis, dislocation of the incudostapedial joint, and bleeding from an exposed jugular bulb. the risks and benefits must be weighed carefully when considering the procedure. myringotomy is an incision into the tm to drain middle ear fluid acutely and usually is preceded by tympanocentesis. indications for myringotomy include treatment for complications of purulent om, such as mastoiditis, labyrinthitis, or facial nerve paralysis. severe otalgia may be relieved by myringotomy. most cases of ome are identified when children return for follow-up after a recent aom. ome also may be an incidental finding. residual fluid in the middle ear may represent incomplete resolution of the acute infection or the natural course of an uncomplicated treated infection. distinguishing between these two entities can be particularly challenging (table 61 -2). figure 61 -5 shows the persistence of mee after a diagnosis of aom. approximately 40% of children continue to have an effusion at 4 weeks, 20% at 8 weeks, and 10% at 12 weeks. frequently these patients are asymptomatic (e.g., no fever, normal hearing), but occasionally children continue to complain of unilateral hearing loss. the tm of ome is retracted (as indicated by a prominent short process of the malleus) with minimal or no signs of inflammation. pneumatic otoscopy reveals diminishedto-absent mobility of the tm. in equivocal cases, tympanometry can be a useful adjunct. the positive predictive value of an abnormal or flat tympanogram is 49% to 99%, which means that half the number of ears with abnormal tests may have ome. a normal tympanogram more accurately predicts absence of an effusion. the development of an intratemporal complication of acute or chronic ear disease should be suspected by clinical signs and symptoms. acute mastoiditis usually presents with fever and postauricular swelling, erythema, and tenderness to palpation. common symptoms associated with aom also may be present, such as otalgia and hearing loss. the pinna is displaced downward and outward, with swelling or sagging of the posterosuperior canal wall (fig. 61-6) . acute otorrhea may be seen as the result of a perforation in the tm. the canal is filled with purulent material and debris. visualization of the tm can be difficult, but it is important to differentiate between aom with a spontaneous perforation and mastoiditis. in such cases, assistance from an otolaryngologist may be needed. rarely a postauricular fistula may develop from the mastoid area, or a fluctuant subperiosteal abscess may be palpated in the same region. children who have persistent retroauricular pain and a history of recurrent om may not have other signs of an acute infection but may have clinically significant disease. the diagnosis of acute and chronic mastoiditis is confirmed by computed tomography of the temporal bone. even in the absence of fluid in the middle ear by otoscopy, any haziness, distortion, or destruction of the bony trabeculae in the mastoid cavity by computed tomography indicates the presence of mastoiditis. these findings should prompt the generalist to seek consultation from an otolaryngologist for assistance in the evaluation and treatment of this condition, especially because a surgical procedure may be indicated. other indications for consultation include a history of recurrent or chronic om, especially if the condition is bilateral, and a concern that the child's hearing, speech, or language development is abnormal. the presence of otorrhea or blood in the external canal suggests the possibility of an acute perforation of the tm or chronic suppurative om with or without cholesteatoma. the challenge for the clinician is to distinguish these two entities from simple oe. specific features of the history and physical examination associated with each condition are summarized in table 61-3. patients with acute oe complain of pruritus, the acute onset of unilateral ear pain that is worsened by applying pressure to the tragus or with movement of the pinna, and sometimes a sense of aural stuffiness or fullness. significant edema of the external canal can cause hearing loss. otalgia may range from mild discomfort to severe, unrelenting pain requiring narcotic analgesics. occasionally, normal jaw movements (chewing) exacerbate the pain. in mild acute disease, the external canal is minimally edematous and erythematous, with or without serous or purulent drainage filling the lumen (box 61-1). as the infection progresses, the exudate becomes more profuse and malodorous. often the pinna and tragus are exquisitely tender to palpation. sagging of the superior canal, periauricular edema, and preauricular and postauricular adenopathy develop with more severe disease. extension of the infection to surrounding soft tissues results in obstruction of the ear canal with or without a circumscribed cellulitis. involvement of the parotid gland, mastoid bone, and infratemporal fossa are other rare complications. traumatic perforations of the tm usually are accompanied by considerable pain and bleeding from the ear that stops spontaneously. a mild conductive hearing loss may be observed on audiometric testing (10 to 35 db), although young children rarely complain of this consequence of injury. an isolated accidental injury to the tm must be differentiated from injury caused by physical abuse. a suspicion of nonaccidental trauma or the presence of other stigmata suggesting child abuse mandates an immediate referral to child protective services and the performance of long bone radiographs. regardless of the etiology, significant complications can accompany traumatic perforations. the presence of a significant sensory hearing loss or severe vertigo indicates a perilymphatic fluid leak into the middle ear space secondary to a transient subluxation of the stapes into the inner ear vestibule. these injuries require a referral to an otolaryngologist for further evaluation and treatment. congenital cholesteatoma usually is discovered incidentally on routine otoscopic examination of an asymptomatic child by the general pediatrician. it appears as a pearly white mass medial to an intact tm, in the anterior-superior quadrant ( fig. 61-7) . a cholesteatoma can be diagnosed during the routine evaluation of a hearing deficit; unilateral hearing loss is detected by audiometric testing. examination with an otomicroscope by a pediatric otolaryngologist confirms the diagnosis. children with acquired cholesteatoma usually have a long history of aom or chronic suppurative om (see fig. 61-7) . occasionally the child is asymptomatic and is noted only to have a retraction pocket in the posterior-superior portion of the tm. more commonly, acquired cholesteatomas are associated with a chronically draining ear and hearing loss or vertigo. mild hearing loss and other subtle symptoms, such as tinnitus, may be missed in children. fever is not a sign of cholesteatoma and if present requires search for another intratemporal or intracranial suppurative complication of middle ear disease, especially when accompanied by otalgia. pneumatic otoscopy is essential to distinguish between tympanosclerosis and a middle ear congenital cholesteatoma. the tm should move independently of a middle ear mass when pressure is applied and released in the external canal. a white tympanosclerotic plaque moves with the rest of the tm. because direct visualization of either type of cholesteatoma is often difficult, general pediatricians should maintain a high index of suspicion when a tm appears abnormal or if a defect in the tm is associated with pearly white squamous debris. adjunctive tests, such as tympanograms and audiograms, are not diagnostic and may be normal even when a lesion is present. all children suspected of having a cholesteatoma should be referred to a pediatric otolaryngologist. high-resolution computed tomography of the temporal bone is helpful when the condition is suspected and often is necessary to delineate the extent of the lesion. the differential diagnosis of hearing loss and indicated diagnostic studies for each condition are discussed in chapter 67. therapeutic goals for infants and young children with om are to treat acute infections appropriately and prevent recurrent and chronic middle ear disease, reducing the morbidity and mortality associated with intratemporal and intracranial complications. another goal is to prevent associated speech and language delay. most cases of aom and ome can be expected to resolve spontaneously. short-term resolution of aom most likely reflects the host immune response and local inflammatory reaction. the primary benefit of antibiotic treatment is reduction of the incidence of delayed suppurative complications. selection of appropriate empirical therapy should be based on its effectiveness against the most predominant organisms. despite the emergence of resistant s. pneumoniae, amoxicillin continues to be standard first-line therapy for uncomplicated aom. this agent is well tolerated by most children, is inexpensive, and maintains an excellent safety record. a dose of 80 to 90 mg/kg/day three times a day for 5 to 10 days is now recommended as empirical initial therapy. the 5-day course can be considered for children 6 years old and older. high-dose amoxicillin is recommended, since the likelihood of highly resistant bacteria is increased in children younger than 24 months of age, children who attend day care, and children who have been treated recently with β-lactam drugs. erythromycin-sulfisoxazole acetyl, clarithromycin, and azithromycin dehydrate are alternatives for children who are allergic to penicillin (type i reaction). failure while on therapy for at least 3 days or relapse within 2 weeks of treatment requires coverage against box 61-1. causes of acute otorrhea ■ aom with spontaneous perforation of the tm ■ aom with transudate on the tm and liquefied cerumen ■ aom with patent tympanostomy tube ■ severe otitis externa ■ trauma of external canal, tm, or temporal bone (blood or cerebrospinal fluid) ■ foreign body with inflammatory reaction ■ contact dermatitis or draining furuncle aom, acute otitis media; tm, tympanic membrane. β-lactamase-producing h. influenzae and resistant strains of s. pneumoniae. antibiotics currently recommended for second-line therapy are those that are effective against these organisms while achieving adequate concentrations in the middle ear. high-dose amoxicillin-clavulanate (90 mg/kg/day of amoxicillin component, with 6.4 mg/kg/ day of clavulanate in two divided doses) and many of the oral cephalosporins (cefuroxime axetil, cefpodoxime, and cefdinir) are second-line therapies. according to a centers for disease control and prevention working group on drugresistant s. pneumoniae, clindamycin might be effective against pneumococci that are resistant to β-lactam antibiotics. a 3-day course of ceftriaxone (50 mg/kg/day), either intravenously or intramuscularly, can be used in children vomiting or in other clinical situations that preclude administration of oral antibiotics. a substantial number of pneumococcal isolates are resistant to erythromycin. therefore, erythromycin-sulfisoxazole is not optimal second-line therapy for aom. the use of trimethoprimsulfamethoxazole as an alternative agent is controversial because pneumococcal surveillance studies indicate that approximately 25% of isolates are resistant. failure of second-line therapy is an indication for tympanocentesis to confirm the diagnosis of om and to obtain a specimen for culture. before this procedure, patient compliance must be ascertained. if good compliance can be confirmed, the infection is most likely due to a resistant strain of pneumococci. the role of intramuscular ceftriaxone in this setting has been investigated and is advocated by most experts. others recommend referral to an otolaryngologist, however, in the case of recurrent disease for placement of tympanostomy tubes. adenoidectomy is reserved for children older than 4 years of age who need tympanostomy tubes a second time. children with acute inflammation of the middle ear may benefit from symptomatic therapy to reduce pain. oral acetaminophen and ibuprofen are acceptable analgesics for most cases of mild-to-moderate pain. topical anesthetics include benzocaine formulations (auralgan and americaine). some families may use home remedies for the treatment of ear discomfort, such as placing a clove of garlic or warm olive oil in the external auditory canal. the role of antihistamines and decongestants in the treatment of acute infections has not been well supported. although these medications may improve symptoms associated with a viral upper respiratory tract infection, studies have not shown that they eradicate middle ear fluid any faster. corticosteroids also have no role in the treatment of aom, although their use remains controversial for long-standing mee. longitudinal studies of ome show that most effusions resolve spontaneously within 3 months. specific recommendations have been developed for young children age 1 through 3 years who are otherwise healthy (e.g., with no craniofacial or neurologic abnormalities or sensory deficits). highlights from a u.s. consensus panel in 1994 include a short period of observation for most children, a hearing evaluation if the effusion is bilateral and present for more than 3 months, control of environmental risk factors such as exposure to tobacco smoke, and antimicrobial therapy for the few children whose effusions persist. because most middle ear effusions resolve without treatment, the general pediatrician should reexamine the asymptomatic child 6 weeks from initial diagnosis with pneumatic otoscopy. if the effusion persists at follow-up, its presence should be confirmed by tympanometry, followed by oral antibiotic therapy for 10 days. for young children that continue to have ome 3 months after the initial diagnosis, a hearing evaluation should be performed and a referral made for possible myringotomy with tympanostomy tube placement, especially if the patient is found to have a hearing deficit. steroid medications, antihistamine/decongestant therapy, and adenoidectomy with or without tonsillectomy are not recommended for the treatment of ome in otherwise healthy children 1 through 3 years old. the reader is referred to the practice guideline published by the american academy of pediatrics for further details regarding the management of ome in young children. adenoidectomy has been shown to be of value in children older than 4 years of age with bilateral effusion. antimicrobial prophylaxis should be reserved for patients with a history of recurrent om and administered during the months when upper respiratory infections are the most prevalent. current treatment options are a single daily dose of amoxicillin, 20 mg/kg, and sulfisoxazole, 75 mg/kg/day divided in two doses. children who continue to have episodes of aom despite medical prophylaxis should be referred to an otolaryngologist for further evaluation and consideration for tympanostomy tube placement. the administration of prophylactic antibiotics for the prevention of mee is not advisable because of bacterial resistance. parental education and the administration of specific immunizations can play a major role in the prevention of aom. as a part of anticipatory guidance, the generalist always should review the role of passive tobacco smoke exposure, bottle-feeding instead of breast-feeding, and pacifier use in the development of aom. additional interventions include providing the influenza vaccine for children who are particularly susceptible to an acute infection during the winter months and administering the heptavalent conjugate pneumococcal vaccine routinely at well-child visits. data report that the pneumococcal vaccine reduces the incidence of aom by 6% to 8%. the management of acute mastoiditis may be medical or surgical, depending on the extent of bony destruction. mastoiditis is an indication for referral to an otolaryngologist. for uncomplicated mastoiditis, intravenous antibiotic therapy is the mainstay of treatment. a myringotomy also may be necessary to decompress the middle ear and is important to provide a specimen for culture and sensitivity. if there is evidence of significant destruction of the bone or no improvement while on intravenous medication, a mastoidectomy is indicated. the goal is to clean out and drain the mastoid air cell system into the middle ear. in addition, a tympanostomy tube is inserted to allow further drainage and ventilation of the middle ear. mild-to-moderate oe can be managed by the general pediatrician with the primary goals of therapy aimed at controlling the inflammation and infection. although difficult and occasionally painful for the patient, the ear canal should be cleansed gently of the debris from the entire length of the external auditory canal. this cleansing may be accomplished by gentle irrigation with warmed saline or 2.5% acetic acid solution. gentle suctioning adequately clears the canal. most cases also require the instillation of ototopical drops into the ear canal or onto a wick if there is significant edema. classes of ototopical medications include steroids, acidifying agents, antiseptics (alcohol), and antibiotics. many of the most commonly used products contain polymyxin b or e, neomycin, and hydrocortisone. the usual dosage schedule is three to four times a day for 7 to 10 days. if a wick is placed, it should be removed within 24 to 72 hours. newer fluoroquinolone topical antibiotics contain ciprofloxacin/hydrocortisone and ofloxacin. patients who do not respond well to initial treatment should be switched to drops containing ciprofloxacin or tobramycin. otomycosis requires treatment with antifungal drops, such as clotrimazole. swimming should be prohibited during the course of treatment. further episodes of oe can be prevented through the use of earplugs while swimming. other recommendations include the use of acidic solutions in the ear canal after exposure to water, drying the ear with a hair dryer set on low heat from 1 foot away for 60 seconds, and avoiding manipulation of the ear canal with cotton swabs and other objects to avoid irritation and maceration of the skin. cases of oe that are persistent or recurrent warrant a culture of the ear discharge and the assistance of a specialist to evaluate the child for cholesteatoma or another undiagnosed condition. traumatic perforations of the tm should be managed in consultation with an otolaryngologist. although small perforations often heal spontaneously within a few weeks after the injury, larger perforations may persist. consultation with a specialist is essential to evaluate the extent of the defect, any associated complications such as involvement of the ossicular chain, and the timing of any surgical procedures. the management of cholesteatoma is surgical and requires the expertise of a pediatric otolaryngologist. the goals of surgery are twofold: (1) to eradicate the squamous epithelium from the middle ear, mastoid, or both and (2) to preserve or restore hearing. the initial surgical procedure is aimed at direct removal of the squamous epithelium. removal is done either through the ear canal or by a postauricular approach, depending on the size of the external auditory canal and location of the lesion. commonly, with acquired cholesteatoma, mastoidectomy also is necessary to remove the entire lesion. other surgical procedures may include ossicular chain reconstruction. hearing aids often are necessary for children who are awaiting elective surgery and have significant bilateral hearing loss or who are not candidates for ossicular chain reconstruction. children with uncomplicated aom generally have a favorable outcome depending on their age at diagnosis, frequency of infections, and length of time for fluid in the middle ear to resolve. severe adverse sequelae of treated om are rare. the most common adverse outcomes include hearing impairment, speech delay, and significant tm perforation. various studies indicate that recurrent disease or prolonged mee in young infants may affect hearing and normal speech and language development. referral to a pediatric otolaryngologist for tympanostomy tube placement is essential for children with severe-to-profound hearing loss or children with abnormal speech development. other children should be evaluated on a case-by-case basis to assess whether medical prophylaxis (antibiotics) should be initiated before the referral. for children who require a second placement of tympanostomy tubes, adenoidectomy without a tonsillectomy has been shown to be an effective additional procedure to prevent recurrent disease. the outcome of intracranial complications of aom, such as meningitis, is variable, ranging from full recovery with no long-term sequelae, to mild hearing loss with minimal associated neurologic deficits, to recurrent seizures, to developmental delay. reported mortality rates in severely affected children range from 0% to 20%. the outcome of intratemporal complications depends on the particular condition being considered. mastoiditis usually has a favorable outcome if the infection is controlled acutely. the outcome of tm perforation depends on the size and location of the defect and any associated complications. most lesions heal spontaneously, however. after an uncomplicated tm perforation, a mild conductive hearing loss may be observed on audiometric testing. destruction of the margin of the tm may lead to additional complications, such as secondary cholesteatoma formation. in children, cholesteatoma is thought to be an aggressive disease that, if not discovered early, may have a poor outcome. because extensive disease usually is found at the time of surgery and there are higher rates of residual and recurrent disease after surgery, the outcome is variable. general recommendations for follow-up of aom are based on several factors, the most important being the amount of time for the accompanying mee to resolve. because approximately 80% of effusions resolve by 8 weeks after the acute infection, most practice guidelines recommend reexamination of the tm within 6 to 8 weeks of the initial diagnosis. audiologic testing usually is reserved for children with a history of recurrent om, chronic mee, or suspected speech delay. an evaluation by a speech therapist is warranted for abnormal language development, especially during the first 4 years of life. a child diagnosed and treated for mastoiditis should continue to be screened for further episodes of om in consultation with an otolaryngologist. children with traumatic perforations should be screened initially for a hearing deficit and then followed until complete resolution of the defect has been documented. all children with cholesteatoma should continue to be followed by a subspecialist so that recurrent lesions can be prevented and controlled. bibliography american academy of pediatrics otitis media guideline panel: managing otitis media with effusion in young children clinical course, complications and sequelae of acute otitis media acute and chronic mastoiditis in children otitis externa recurrent and persistent otitis media disorders of the eye may be congenital, developmental, hereditary, infectious, immune-mediated, traumatic, neoplastic, and neurologic conditions. ophthalmic manifestations may be the heralding sign in a spectrum of systemic conditions, such as stevens-johnson syndrome, crouzon's disease, or albinism, in which the eye may be the most significantly involved structure. an understanding of both neurologic function and the basic function and anatomy of the eye, periocular structures, and orbit is essential when developing an assessment and treatment plan.conditions that affect the eye or its function often are subdivided by the particular ocular structure involved. different disorders can lead to a final common pathway of dysfunction. treatment of the presenting condition requires evaluation of a broad differential diagnosis and the effect of the condition on the developing visual system.the ocular system is subclassified by the ocular structures. external disorders are conditions that affect the orbit, lids, or lacrimal system. disorders of the anterior segment are conditions that affect the cornea, iris, or lens or lead to glaucoma. posterior segment disorders involve the retina, retinal vasculature, vitreous humor, and optic nerves. neuroophthalmologic disorders, inflammatory conditions, and trauma often involve multiple structures. vision development, nystagmus, amblyopia, and ocular torticollis are special ocular conditions that affect pediatric patients. figure 62 -1 depicts the important internal anatomic structures of the eye. each structure is highly specialized to provide an enormous range of information to the visual system. the eye is responsible for discrimination and resolution (visual acuity), sensing object movement, peripheral vision, and positional visual awareness. the visual system is able to adjust over a range of about 10 log units in the ambient light intensity levels. the tracking mechanisms of the eye also are finely developed to provide for image stabilization and pursuit and rapid and directed (saccadic) eye movements that allow for refixation and rapid response to new objects of regard in the visual awareness. the cornea, the most anterior structure of the eye, is composed of five layers: the epithelium and its basement membrane, the stroma, and the endothelium and its basement membrane. the junction of the clear cornea and the white sclera is known as the corneal-scleral limbus and is an important anatomic landmark. the epithelium, a unique key: cord-322760-tsxniu3j authors: sha, jianping; li, yuan; chen, xiaowen; hu, yan; ren, yajin; geng, xingyi; zhang, zhiruo; liu, shelan title: fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea date: 2016-09-23 journal: arch virol doi: 10.1007/s00705-016-3062-x sha: doc_id: 322760 cord_uid: tsxniu3j middle east respiratory syndrome coronavirus (mers-cov) was first isolated in 2012. the largest known outbreak outside the middle east occurred in south korea in 2015. as of 29 june 2016, 1769 laboratory-confirmed cases (630 deaths; 35.6 % case fatality rate [cfr]) had been reported from 26 countries, particularly in the middle east. however, the cfr for hospital outbreaks was higher than that of family clusters in the middle east and korea. here, we compared the mortality rates for 51 nosocomial outbreaks in the middle east and one outbreak of mers-cov in south korea. our findings showed the cfr in the middle east was much higher than that in south korea (25.9 % [56/216] vs. 13.8 % [24/174], p = 0.003). infected individuals who died were, on average, older than those who survived in both the middle east (64 years [25–98] vs. 46 years [2–85], p = 0.000) and south korea (68 years [49–82] vs. 53.5 years [16–87], p = 0.000). similarly, the co-morbidity rates for the fatal cases were statistically higher than for the nonfatal cases in both the middle east (64.3 % [36/56] vs. 28.1 % [45/160], p = 0.000) and south korea (45.8 % [11/24] vs. 12.0 % [18/150], p = 0.000). the median number of days from onset to confirmation of infection in the fatal cases was longer than that for survivors from the middle east (8 days [1–47] vs. 4 days [0–14], p = 0.009). thus, older age, pre-existing concurrent diseases, and delayed confirmation increase the odds of a fatal outcome in nosocomial mers-cov outbreaks in the middle east and south korea. electronic supplementary material: the online version of this article (doi:10.1007/s00705-016-3062-x) contains supplementary material, which is available to authorized users. the first report of middle east respiratory syndrome (mers) was identified in saudi arabia in june 2012. the middle east respiratory syndrome coronavirus (mers-cov) isolated from this patient was similar to severe acute respiratory syndrome coronavirus (sars-cov), which caused an epidemic in 2002-2003 [49] . both novel viruses are single-stranded rna viruses belonging to the genus betacoronavirus [25, 48] , and the diseases they cause share common clinical characteristics, including fever, cough, diarrhea, and shortness of breath [5] . jianping sha, yuan li, and xiaowen chen equally contributed to this work. as of 29 june 2016, the world health organization (who) had been notified of 1769 laboratory-confirmed cases with mers-cov (globally), including at least 630 deaths; the case fatality rate (cfr) was 35.6 % (630/1769) [46] . a total of 26 countries in the world have been affected, including countries in the middle east (egypt, iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates, yemen), africa (algeria, tunisia), europe (austria, france, germany, greece, italy, the netherlands, turkey, the united kingdom), asia (china, the republic of korea, malaysia, philippines, thailand) and north america (united states) (http://www.who.int/ emergencies/mers-cov/en/). so far, all cases of mers have been linked through travel to or residence in countries in or near the middle east. generally, the middle east is the primary region where mers-cov originates, circulates and is exported. in contrast, since the first report of sars-cov in china in 2003, a total of 8096 sars cases, including 774 deaths, have been reported to who. these have involved 19 countries, predominantly in south east asia, with only one case identified in kuwait in 2003, and no cases were found in the middle east since then (http:// www.who.int/csr/sars/country/table2004_04_21/en/). the fatality risk for mers-cov is much higher than that for sars-cov, which has a cfr of 9.6 % [9, 24] . furthermore, the cfr for patients with co-morbidities is greater (60 % in mers vs. 46 % in sars) than those without preexisting diseases [49] . generally, the cfr is attributed to both host factors and virus factors (e.g. virus replication and mutation) and local medical expertise [3, 14] . one unique common epidemiological characteristic of these two diseases is that the spread of both mers-cov and sars-cov infection has been largely driven by human-to-human transmission in healthcare settings [25] . failures in infection prevention and control in healthcare settings have occasionally resulted in large numbers of secondary cases in nosocomial outbreaks. the earliest identified nosocomial mers outbreak was traced back to march 2012 from clusters of severe respiratory illness among healthcare personnel (hcp) in jordan [43] . since then, a series of nosocomial mers outbreaks in small numbers have been identified in the middle east (jordan in 2012, saudi arabia in 2014-2015) [1, 6, 10, 18, 36] in this study, we conducted a preliminary mortality risk factor analysis for nosocomial mers-cov outbreaks in south korea and the middle east. the findings from this study might help to reduce the severity and number of deaths from hospital-clustered cases by leading to the adoption of appropriate control measures. in 2015, scientists in the republic of korea and china completed full-genome sequencing of coronaviruses from the mers outbreak in korea. the findings were analysed by a group of virologists convened by who, and preliminary results suggested that the mers cov viruses isolated in the republic of korea were similar to those isolated in the middle east (http://www.who.int/mediacentre/news/ mers/briefing-notes/update-15-06-2015/en/). mers-covs associated with the korean and middle east outbreak belong to lineage 5 of mers-cov, which has been the predominant infectious agent in saudi arabian camels since november 2014 [41] . the mers-cov variants associated with the recent outbreak of human infections in south korea (e.g., chinagd01-v1/2015 and kor/knih/ 002-05/2015) show the highest similarity (99.96-99.98 %, full genome) to a camel virus (camel/riyadh/ry159/2015) sampled in march 2015, followed by the latest strain (kt026453) prevalent in saudi arabia (99.92 % identified) [26] . however, the mers-covs in korea have the ability to cause large outbreaks in environments that are different from that of the middle east (http://www.who.int/emer gencies/mers-cov/en/). the national health and family planning commission of china determined that the collection of data from one human mers-cov infection imported from korea was part of the public health investigation of an outbreak and was exempt from institutional review board assessment. all other mers cases were obtained from publicly available data sources. all data were supplied and analysed without access to personal identifying information. information on all laboratory-confirmed mers cases was obtained from various publicly available sources, including who global alert and response updates, documents officially released by the local health bureau, news releases from middle eastern and south korean authorities, the weekly epidemiological record, promed posts, and literature published from 1 april 2012 to 29 june 2016 (http:// www.who.int/csr/don/archive/disease/coronavirus_infections/ en/). the latest cases that had not been officially announced by who were identified by searching promed posts, which confirmed announcements by individual countries' ministries of health. based on the available data, we initially established a database of a line list of human nosocomial mers outbreaks (supplementary tables s1, s2 and s3). according to the who's 14 july 2015 interim reporting definition (http://www.who.int/csr/disease/coronavirus_ infections/case_definition/en/), a person with mers has a laboratory-confirmed mers-cov infection, irrespective of clinical signs or symptoms. a case may be laboratoryconfirmed by detection of viral nucleic acid or serology. the presence of viral nucleic acid can be confirmed by either a positive reverse transcription polymerase chain reaction (rt-pcr) result on at least two specific genomic targets or a single positive target with sequencing of a second target. a case confirmed by serology requires demonstration of seroconversion in two samples, ideally taken at least 14 days apart, by enzyme-linked immunosorbent assay (elisa), by indirect fluorescent antibody (ifa) screening, or by a neutralization assay [25, 49] . a direct epidemiological link with a confirmed mers-cov patient may include (1) healthcare-associated exposure, including providing direct care for mers-cov patients, working with healthcare workers infected with mers-cov, visiting patients or staying in the same close environment of individuals infected with mers-cov; (2) working together in close proximity or sharing the same classroom environment with individuals infected with mers-cov; or (3) travelling together with individuals infected with mers-cov in any kind of conveyance or living in the same household as individuals infected with mers-cov. in addition, the epidemiological link may have occurred within a 14-day period before or after the onset of illness in the case under consideration [25] . we used a comparative epidemical analysis of the dates of onset of illness and the characteristics of the fatal and surviving cases. all statistical analysis was conducted using the statistical analysis system, version 9.2 (sas institute, cary, nc, usa). quantitative measurements are presented as the median and range of the observed values, and qualitative measurements are presented as relative and absolute frequencies. an analysis of variance (f test) was applied to the measurement data. v 2 tests were used to compare the distribution of the different variables of qualitative measurements between fatalities and survivors. fisher's exact test was used in the analysis of contingency tables when the sample sizes were small (the expected values in any of the cells of a contingency table were below 5; the number of total samples was no more than 40; the data were very unequally distributed among the cells of the table). any p-values given were two-sided and considered statistically significant at 0.05. as of 31 march 2016, we had identified 47 human laboratory-confirmed clusters with mers-cov, involving 179 cases, of which 53 were fatal. all clusters had been reported to who or published by the local authority or in pubmed. these mers-clustered cases were distributed in nine countries: 29 clusters from the kingdom of saudi arabia (ksa), six from the united arab emirates (uae), four from jordan, three from qatar, and one each from france, iran, italy, tunisia, and the united kingdom (uk). the numbers of clusters per year were as follows: three clusters including 18 cases in 2012, 33 clusters including 108 cases in 2014, and 11 clusters including 53 cases in 2014. for the control groups, we chose a total of 504 sporadic cases of mers-cov, composed of 129 fatal and 375 nonfatal cases from the following countries: 350 cases from the ksa, 125 cases from the uae, 10 cases from jordan, 10 from qatar and 9 from tunisia. the numbers of sporadic cases per year were as follows: 110 cases in 2012, 350 cases in 2013 and 44 cases in 2014. fatality risks for nosocomial mers outbreaks 35 the results showed that the percentages of hcp in mers clusters were much higher than those in sporadic cases (32.4 % [58/179] vs. 10.7 % [54/504], p = 0.000) ( table 1 and table s1 ). however, the hcp-specific cfr was much lower than the overall cfr from mers clusters ( , p = 0.000). the median age in fatal cases in hcp was much lower than in fatal cases overall (46.5 years vs. 57 years , p = 0.000) ( table 1) . we stratified the age groups between the fatal and nonfatal cases groups. the results showed a statistical difference in the distribution of the 0-14, 15-29, 30-44, 45-59, and 60? year-olds between the two groups (p = 0.000). males dominated both the fatal and nonfatal groups of the clustered and sporadic cases (p [ 0.05) ( table 1) . a history of exposure to camels prior to onset of disease was not significantly correlated with survival (7. five time periods useful for public health surveillance were evaluated. the median time from onset to confirmation of infection in the fatal groups was much longer than that for survivors in mers clusters (12.5 days [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] vs. 9 days [0-24], p = 0.041) and in sporadic mers cases (12 days vs. 9 days [0-30], p = 0.003). however, there were no statistical differences in the median time from onset to hospital admission, onset to hospital discharge, and subsequent death or the number of hospitalized days between the fatal and nonfatal cases for the two groups (table 1) . by 30 march 2016, we had obtained data on 51 nosocomial outbreaks involved in 216 confirmed cases (all 51 nosocomial outbreaks were from the middle east; the above 47 clusters were not included in these outbreaks), including iran (one cluster), ksa (41 clusters), jordan (three clusters), france (one cluster) and uae (five clusters). we also had one nosocomial outbreak with 174 confirmed cases with mers-cov in south korea (table 2 and table s2 ). the observed average cluster size (174) for mers from south korea was much greater than that for the middle east (4, range 2-28). human nosocomial outbreaks with mers-cov in the middle east occur throughout the year and peak in the spring, especially february to april. mers outbreaks in south korea were reported from march to june 2015, concomitant with peaks in the reporting of mers nosocomial outbreaks in the middle east ( table 2) . the average age in the fatal groups was much higher than that in the survival groups (64 years old vs. table 2) . we found no difference between the fatal and nonfatal cases with respect to exposure to camels and other animals (horses, sheep and goats). in contrast, the level of humanhuman transmission was much higher in the nonfatal cases in the middle east than in the fatal cases (86. table 2) . the middle east group showed a statistical difference between fatal and nonfatal cases for the median days from 165], p = 0.000). however, there were no differences in the percentage of total deaths between the index and secondary cases ( table 3) . the mean age of the deaths was significantly higher than that of the survival cases for the index (64 years vs. 54 years [24-85, p = 0.038) and secondary cases (43 years vs. 37 years , p = 0.030). patients in the age groups c60 and 45-59 years were the most common in the fatal and survival cases, respectively, for the index group, while the 45-59 and 30-44-year age groups were the common groups in the fatal and nonfatal cases, respectively, for the secondary cases. there was no significant difference in gender distribution between the fatal and nonfatal cases in the index and secondary groups ( table 3) . the ratio of co-morbidity was much higher in the fatal groups than in the non-fatal groups from the secondary cases (37.5 % [12/32] vs. 17.1 % [19/111], p = 0.026); however, there was no difference between the fatal and nonfatal groups from the index cases. similarly, a history of exposure prior to onset was common for the fatal and nonfatal groups from the index and secondary cases ( table 3) . there were no differences between fatal and nonfatal cases in the median time from onset to hospitalization, onset to confirmation, onset to discharge or death or hospitalized days (table 3) . however, the median time from onset to hospitalization was shorter in the secondary cases compared to the index cases (3 days the secondary cases was slightly shorter than in the index cases (9 days vs. 14 days , p = 0.033); however, the median time from onset to hospital discharge for secondary survivors was 10 days (6-18), which was significantly shorter than the 14 days (3-31) for index survivors (p = 0.025). acute respiratory tract infections with mers-cov cause considerable morbidity and mortality and pose a threat of repeated outbreaks in healthcare facilities [1, 6, 10, [18] [19] [20] 38] . the resulting transmission among patients, visitors, and hcp has been a defining feature of mers-cov epidemiology since its emergence in 2012 [7] . in this study, we compared the mortality risk factors in two different nosocomial outbreaks, based on 51 nosocomial outbreaks of mers-cov infection in the middle east and one large outbreak identified in south korea. our findings showed the final cfr for the middle east (25.9 %) was significantly higher than that for south korea (13.8 %). both estimated cfrs were significantly lower than that for one hospital outbreak of mers (cfr 65 % [15/23] ) in saudi arabia in 2013 and another nosocomial outbreak (cfr 36.5 % [93/255]) in saudi arabia 2014 [5, 36] . the cfr of this latter outbreak was also much higher than that of one extended family cluster (10.5 % [2/ 19]) in saudi arabia in 2014 [4] . these results demonstrate that the survival rate of clustered patients with mers-cov in korea was higher than in the middle east. there are several possible explanations for the observed differences between the cfrs in south korea and the middle east. first, there may be disparities in national surveillance and available expertise [30] . second, the cfr for the middle east might have been overestimated because a large number of mild and asymptomatic cases are likely to go undetected there [37] . third, it is possible that primary cases accounted for a higher percentage of the cluster patients in the middle east than in south korea [36] . the findings on age were consistent in hospital outbreaks in the middle east and from south korea. our results showed that the median age in fatal cases was much higher than that in nonfatal cases. this is in agreement with a saudi arabian case series report that showed individuals older than 65 years had a greater association with mortality. a multivariate logistic regression model estimated that for every 1-year increase in age, the odds of dying increased by 12 % [29] . in all, this indicates that older age is associated with death in cases of mers-cov infection [12, 17, 44] . in particular, the median age in fatal hcp cases was also much higher than that in nonfatal hcp cases, but lower than the overall average. this is in agreement with the findings of liu et al. [25] . the reasons for the higher fatality rates in older individuals are not understood but have been attributed to cultural practices that result in an increase in the exposure risk that older people are willing to take [37] . in addition, older people may be more likely to smoke and to have underlying diseases and impaired immune functions, which may increase susceptibility and progression of infections and even increase the chance of death [45] . the sex characteristics of mers outbreaks in the middle east are similar to those in south korea. the patients in mers outbreaks in both areas were predominantly male, and the proportion of males in the study populations did not differ [25] . furthermore, there was no difference in the male-specific cfr between the mers clusters of the two groups, a finding that is similar to other reports [1, 2, 10, 18] . our findings suggest that the gender distribution is not linked to a fatal risk factor in mers outbreaks. hcp are at high risk of acquiring emerging mers infections due to occupational exposure and are affected mostly by nosocomial outbreaks [1, 6, 15, 28, 35] [7] . in total, the fatality risk for hcp was significantly lower than the overall fatality risk in the middle east and south korea. these findings can be attributed to three facts: first, the majority of hcp developed asymptomatic or mild symptoms and moderate symptoms [15] ; second, hcp were confirmed as secondary cases under medical investigation, which led to earlier confirmation and good outcomes [32] ; third, epidemiological analysis showed that hcp were much younger and had fewer co-morbidities compared to total mers cases [36] . in contrast with sars, about 75 % of patients with mers had at least one additional illness, and patients who died were more likely to have an underlying condition (86 % of patients who died vs. 42 % of recovered or asymptomatic patients) [47, 49] . similar to the middle east, this study showed that the odds of dying were four times higher for individuals with concurrent health conditions than for those without these conditions in south korea. the odds of fatality were much lower than those estimated by the logistic regression model (seven times) [29] . this is in part due to higher viral loads in the respiratory tract and longer shedding in patients with underlying diseases compared to cases without co-mortalities [33, 49] . human-to-human transmission of mers-cov has been confirmed by epidemiological and genomic studies of cases associated with hospital and household mers outbreaks [13] . spread was assumed to occur largely via large droplets and contact [36] . our study indicated that the percentage of human-to-human transmission in nonfatal cases was slightly higher (92.9 % vs. 64.2 %) than in fatal cases in mers clusters, and two reasons could explain this: first, the survivors in secondary cases were younger and had fewer co-morbidities [11, 19, 20, 29, 38] ; second, most of the secondary cases were under medical investigation, and therefore, the infection could be confirmed early once symptoms were observed, making timely treatment possible [16, 19, 20, 23, 36, 39, 42] . overall, human-to-human transmission seems to have had a positive effect on the outcome of the secondary cases from the mers nosocomial outbreaks in the middle east. rapid diagnosis and providing supportive care may be of marginal consequence in the mers clusters [25, 29] . the progression of illness in fatal and nonfatal infections in nosocomial outbreaks with mers-cov in the middle east does not follow the typical pattern of south korea infections [29] . in the middle east, the median time from onset to confirmation in fatal cases (8 days) was clearly longer than in nonfatal cases (4 days). in south korea, however, there was no difference in the median time between fatal and nonfatal cases. this is consistent with other retrospective studies of mers virus infections [6, 30, 36] . furthermore, the time between suspected symptom onset and laboratory confirmation (6.5 days) in the fatal clusters was also slightly longer than the overall average [38] . in particular, this finding indicated that delayed confirmation is a high-risk factor for human nosocomial outbreaks with mers-cov in the middle east. in conclusion, the overall cfr for nosocomial outbreaks in the middle east was much higher than in south korea. however, the mortality risk factors for mers infections in the middle east were similar to those identified for nosocomial outbreaks in south korea. older age, underlying diseases and delayed confirmation were the major risk factors for fatal outcome in human nosocomial outbreaks. in contrast, person-to-person transmission was associated with a good outcome for secondary cases during nosocomial outbreaks. interestingly, gender, exposure history and median days were not indicators of death with mers nosocomial outbreaks. the severity of nosocomial outbreaks and the risk of fatal infection in hcp were significantly lower than the overall rate in the middle east and south korea. nosocomial outbreaks of mers-cov infection are associated with knowledge deficits, unrecognized disease, insufficient infection control measures, poor compliance, and an overwhelming number of patient cases [21, 22, 34, 40, 45] . therefore, early and rapid detection of suspected cases, especially in older people and hcp, along with appropriate infection control practices, education and timely preparedness, are important strategies to reduce nosocomial transmission and to improve the clinical outcome in health settings in the future [8, 27, 31, 35] . hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome 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laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia association of higher mers-cov virus load with severe disease and death middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling fatal outcome of sars in a patient with reactivation of chronic hepatitis b middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia mers outbreak in korea: hospital-to-hospital transmission epidemiologic features of the first mers outbreak in korea: focus nosocomial transmission of sars co-circulation of human metapneumovirus and sarsassociated coronavirus during a major nosocomial sars outbreak in hong kong middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak risk factors for sars-related deaths in 2003 comparative epidemiology of human infections with middle east respiratory syndrome and severe acute respiratory syndrome coronaviruses among healthcare personnel complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus infection control: the missing piece? screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome middle east respiratory syndrome corona virus, mers-cov middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications mers-cov outbreak in jeddah-a link to health care facilities a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case epidemiological investigation of mers-cov spread in a single hospital in south korea middle east respiratory syndrome-advancing the public health and research agenda on mers-lessons from the south korea outbreak factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the mers-cov outbreak: challenges facing the dental profession risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu fatal aspergillosis in a patient with sars who was treated with corticosteroids critical role of nosocomial transmission in the toronto sars outbreak fatality risks for nosocomial mers outbreaks 43 middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome acknowledgements we thank the reporting countries with the confirmed mers cases. we appreciate their assistance with field investigations, administration and data collection and sending the data to who. conflict of interest none declared. key: cord-343302-g9vcchrh authors: agrawal, anurodh shankar; ying, tianlei; tao, xinrong; garron, tania; algaissi, abdullah; wang, yanping; wang, lili; peng, bi-hung; jiang, shibo; dimitrov, dimiter s.; tseng, chien-te k. title: passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection date: 2016-08-19 journal: sci rep doi: 10.1038/srep31629 sha: doc_id: 343302 cord_uid: g9vcchrh middle east respiratory syndrome coronavirus (mers-cov) has repeatedly caused outbreaks in the arabian peninsula. to date, no approved medical countermeasures (mcm) are available to combat mers-cov infections. several neutralizing human monoclonal antibodies (mabs), including m336, a germline-like human mab, have been chosen as promising mcm for mers-cov. however, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. we assessed the prophylactic and therapeutic efficacy of m336 by using well-characterized transgenic mice shown to be highly sensitive to mers-cov infection and disease. we found that mice treated with m336 prior to or post lethal mers-cov challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. taken together, these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for mers-cov infection. scientific reports | 6:31629 | doi: 10.1038/srep31629 and long-term threat to people, particularly those who interact closely with camels in the arabian peninsula. even though mers-cov presently has limited human-to-human transmission 2, 16 , the high mortality rate of this virus and limited information on the mechanism able to confer increased human-to-human transmission have raised concerns of a potential mers pandemic. indeed, the recent outbreaks in korea and the appearance of super-spreading events indicate that mers-cov has the ability to cause large outbreaks outside of the arabian peninsula [17] [18] [19] . currently, no approved vaccines or drugs are available to treat this viral infection. these facts highlight an urgent need to develop potent prophylactic and therapeutic agents to fight this lethal virus. similar to other coronaviruses, mers-cov uses the envelope spike (s) glycoprotein, a class i transmembrane protein, for interaction with its cellular receptor for binding, fusion and entry into the target cell 20 . the receptor binding domain (rbd) located in the s1 domain of the mers-cov spike is responsible for binding to the well-characterized cellular receptor identified as dpp4 (cd26) and is, therefore, critical for binding and entry of the virus [20] [21] [22] . therefore, neutralizing antibodies capable of blocking such interaction could be promising preventive and/or therapeutic candidates. recently, human monoclonal antibodies (mabs) capable of neutralizing mers-cov have been identified and characterized by several research groups [23] [24] [25] [26] [27] [28] . these antibodies have been isolated from naive human antibody libraries, from transgenic "humanized" mice, or from b cells of an infected individual, and they recognize different epitopes on mers-cov rbd. one of the most potent mabs, m336, is a germline-like antibody identified from a very large (~10 11 size) phage-displayed antibody library derived from b cells of healthy donors. this mab exhibits exceptionally potent neutralizing activity (ic 50 = 0.005 μ g/ml) in vitro 23 . moreover, because its epitope almost completely (~90%) overlaps with the receptor-binding site of dpp4 on mers-cov rbd, as is evident by its recently solved crystal structure 29 , the probability of generation of resistant mutants may be absent or very low. notably, although the functions of these mabs have been extensively characterized in vitro, their further clinical development has been hindered by the lack of an effective animal model of mers-cov infection. mers-cov cannot infect small laboratory animals (e.g., mice, hamsters and ferrets) as a consequence of species-specific differences in dpp4, while only causing mild-to-moderate symptoms in rhesus macaques. marmosets, which are more susceptible to mers-cov, developed a moderate-to-severe disease, but limited availability and high cost have hampered their use 30 . rabbits can be infected, but the infectious virus is challenging to detect 31, 32 . it was found that the expression of human dpp4 could overcome the lack of susceptibility in normal mice. with prior transduction of adenoviral human dpp4-expressing vectors, mice became susceptible to mers-cov infection without revealing any measurable clinical manifestations 33 . in contrast, transgenic (tg) mice with the human dpp4 gene integrated into the genome readily developed acute morbidity (weight loss), and uniform death occurred within a week 34, 35 , making it an ideal preclinical model for the development of vaccines and treatments against mers. some of the aforementioned human neutralizing monoclonal antibodies have been shown to protect engineered human dpp4-expressing mice and the naturally permissive rabbits, entirely based on their ability to inhibit mers-cov infection and/or alleviate histopathology of the lungs 27, 28, 36, 37 . to further verify the protective efficacy of these human monoclonal antibodies, particularly m336, against mers-cov infection, it is highly desirable to use the well-characterized human dpp4 tg mice known to result in acute disease (weight loss) and death 34, 35 . by using this highly permissive tg mouse model, we evaluated the prophylactic and therapeutic efficacy of m336 mab in vivo. we report in this study for the first time that treatment of tg mice with a single-dose of m336 antibody prior to or after challenging with 1,000 ld 50 of mers-cov protected mice from the lethality in a dose-dependent manner, thereby representing the first antibody tested for its protective efficacy against lethal mers-cov infection. prophylactic efficacy of mers-cov rbd-specific human monoclonal antibody, m336. we established a tg mouse model which is profoundly sensitive and susceptible to mers-cov infection, as determined by high viral titers in the lungs, as well as a high rate of morbidity and mortality 34, 38 . equipped with this small animal model of human mers-cov, we investigated the protective efficacy of mab m336. to accomplish this, each group (n = 6) of mice was treated via the intraperitoneal (i.p.) route with two different doses: 0.1 mg and 1 mg per mouse diluted in 100 μ l pbs, and challenged intranasally (i.n.) at 12 h post treatment with 10 4 tcid 50 (i.e., 1,000 ld 50 ) of mers-cov in a volume of 60 μ l 38 . challenged mice were monitored daily for clinical manifestations (weight loss) and mortality. as shown in fig. 1 , the group treated with 1 mg mab survived viral infection without showing any clinical symptoms. these mice initially showed either no weight loss or recovered from mild weight loss within three days (fig. 1a) . on the other hand, the group treated with 0.1 mg mab showed a gradual weight loss (15-20%) until day 13 just before starting to recover (fig. 1a ). all surviving mice (one died on day 13 in mice treated with 0.1 mg of m336) continued to recover and appeared well up to 21 dpi when the experiment was terminated (fig. 1b) . all mers-cov-challenged mice pretreated with a high dose (1 mg) of irrelevant mab m102.4 exhibited profound weight loss (> 15%) and succumbed to infection with 100% mortality by day 8 p.i. (fig. 1a,b ). therapeutic efficacy of mers-cov rbd-specific human monoclonal antibody, m336. to determine the therapeutic potential of this human monoclonal m336 antibody, groups of mice (n = 6 per group) were challenged (i.n.) with 10 4 tcid 50 of mers-cov (i.e., 1,000 ld 50 ) in a volume of 60 μ l and then treated (i.p.) 12 hours later with a single dose of either 1 mg or 0.1 mg of m336 or 1 mg of m102.4 antibody (control) in 100 μ l per mouse, followed by monitoring daily for wellbeing (weight loss and other clinical manifestations) and mortality of mice. we noted that whereas treatment with 1 mg of m336 antibodies was effective in the protection against the lethality caused by mers-cov infection, it failed to protect mice fully from the onset of clinical illness (weight loss). specifically, all of the challenged mice treated with 1 mg of m336 antibody suffered an attenuated (< 10%), and transient weight loss until day 9, and gradually recovered to day 21 when the experiment was terminated (fig. 2) . similarly, challenged mice treated with a low dose of 0.1 mg of m336 antibodies suffered from attenuated and transient weight loss until day 7 p.i. and gradually recovered. however, we noted a single death at day 9 in this low dose treatment group (fig. 2) . as expected, all mice treated with a single dose of 1 mg of control m102.4 antibody exhibited profound weight loss (> 15%) and succumbed to mers-cov infection with 100% mortality by day 8 p.i. (fig. 2) . taken together, these results indicate that this mers-cov rbd-specific human m336 antibody can be highly effective as prophylactic or therapeutic modalities in protecting highly permissive transgenic mice against mers-cov infection and disease. we also investigated the protective mechanism of m336 against mers-cov by determining the lung virus titers in challenged mice at day 2 after treatment. specifically, we sacrificed two mice (out of 6) in each group, as described above for figs 1 and 2 and their lung specimens were harvested for determining viral titers by using via vero e6 cell-based infectivity assay and quantitative pcr (q-pcr)-based assay targeting the upstream e gene of mers-cov. as shown in fig. 3a we were unable to recover infectious virus from any mouse treated with 1 mg of m336 antibody either before or after challenge with mers-cov. however, we were able to detect a barely detectable infectious virus, with the limit of detection (lod) of 2.3 log ticd 50 /g, from a single mouse receiving 0.1 mg of m336 prior to viral challenge. these results indicated that mab m336 most likely confers protection from lethal challenge by restricting viral replication within the lungs, thereby preventing viral infection in the brains and other organs. titers of viral rna copy number, as shown by qrt-pcr assays, were also compared among groups having different doses of mabs. lungs of infected mice were harvested on day 2 post-and pre-virus challenge group. all groups exhibited detectable viral rna. titers were significantly lower than those in the control group in all m336-treated groups. in the pretreatment group, mice treated with 1 mg of m336 showed a 2-log reduction in viral rna detection, while a ~1 log reduction in viral numbers was seen in mice treated within 0.1 mg m336 when compared to mice receiving control mab m102.4. in the post-treatment group, a smaller (~1 log) difference in viral rna copy number (compared to that in the pretreatment group) was observed between mice treated with 1 mg antibody compared with those receiving control antibody, while a more than 1 log reduction in viral rna number was seen in mice treated with 0.1 mg m336 when compared to mice receiving control mab (fig. 3b) . these data indicate that m336 confers significant protection to mice when administered pre-or post-viral challenge. taken together, these results suggest to us that the epitope targeted by this exceptionally potent rbd-specific m336 antibody has a great potential for further development as a potent preventive and therapeutic agent in the future. treatment with m336 attenuates lung pathology associated with mers-cov infection. the effect of m336 antibody treatment on the pulmonary pathology associated with mers-cov infection was evaluated by using formalin-fixed, paraffin embedded, and hematoxylin/eosin (h&e)-stained lung specimens harvested at day 2 p.i. pulmonary pathology was noted in all mice that were treated with different doses of m336 or control m102.4 antibodies either before or after viral infection. on a severity scale of 0 to 3 (none, mild, moderate, severe), h&e-stained samples from mice pretreated with 1 mg and 0.1 mg of m336 antibody were graded 0 and 1, respectively, for perivascular and intra-alveolar infiltration of mononuclear cells, including lymphocytes, macrophages/monocytes (fig. 4, middle panel) , whereas those obtained from mice that received post-infection lung specimens collected at day 2 after viral challenge were processed for assessing the viral titers by using both vero e6-based infectivity assay and qrt-pcr targeting upstream e gene of mers-cov, and expressed as log 10 tcid 50 /gram and log 10 tcid 50 equivalent (eq.)/gram, respectively. (a) prophylactic and therapeutic efficacy of human m336 antibody treatment in reducing the lung titers of infectious virus. (b) prophylactic and therapeutic efficacy of human m336 antibody in reducing the titers of viral rna. the data shown are representative of at least two independently conducted assays using the same samples. data is presented as mean ± standard error (se). * * * p < 0.001 as determined by using student's t test. scientific reports | 6:31629 | doi: 10.1038/srep31629 treatment with either dose of m336 were graded 1 (fig. 4, right panel) , compared to the grade 2 assigned to mice received control antibody treatment prior to infection (fig. 4, left panel) . mers-cov has attracted significant basic research and clinical studies since it was first discovered in early 2012. even though the transmissibility of mers-cov among humans remains low at present, as a mutation-prone rna virus, it could eventually evolve into a highly communicable and more virulent human pathogens. this emphasizes the urgent need for the development of an effective antiviral therapy which could restrict the spread of this deadly disease. in other viral infections, neutralizing antibodies have been shown to protect the host from disease progression and/or reduce the severity of clinical symptoms. passive immunotherapy for prophylaxis and treatment of infectious viral diseases has been widely used for many decades [39] [40] [41] [42] [43] . passive transfer of neutralizing antibodies is also a promising strategy for both prophylaxis and treatment against mers-cov infection. to this end, we and others have successfully demonstrated the protective efficacy of specific human neutralizing monoclonal antibodies in animal models of mers-cov infection 23, 24, 26, 28 . among a panel of mers-cov-specific mabs generated by using a vast phage display library 23 , we identified three mabs which specifically bind to the mers-cov rbd with very high affinity. among these three identified, we noted that mab m336 exhibited the highest potency in neutralizing live mers-cov. here, we further characterized this novel human mab in our tg mouse model of mers-cov infection and showed prophylactic and therapeutic protection of mice treated with m336 before and after a lethal challenge with the virus, respectively. thus, mab m336 is highly promising as a potent inhibitor for urgent prophylaxis in adjunctive treatment for patients infected with mers-cov. in our studies, we noted that passively transferred with 1 mg and 0.1 mg of m336 monoclonal antibodies to individual mice 12 h prior to challenge with 1,000 ld 50 of mers-cov resulted in 100% and 75% protection against lethality, respectively (fig. 1) , suggesting that using 0.1 mg m336/mouse as a prophylaxis is suboptimal to completely neutralize viral infection, thereby allowing residual viruses to replicate within lungs during the course of infection. these data demonstrate that m336 confers a dose-dependent reduction of mers-cov infection, corroborating lower viral rna levels and live virus isolation determined for these mice when compared to control mice. our study also confirmed the therapeutic efficacy of m336 in a dose-dependent manner. similar to the prophylactic studies, administration of a single-dose of m336 antibody at a concentration of either 1 or 0.1 mg per mouse at 12 h after mers-cov challenge provided 100% and 75% protection, respectively, against infection-induced lethality, accompanied by reduced viral loads (both infectious virus and viral rna) within the lungs. however, we also noted the recovery of bodyweight loss and the reduction of viral loads in mice treated with 1 mg of m336 at 12 hrs after infection were slower than those treated with 0.1 mg of m336, as shown in figs 2a and 3b, respectively. while there is no clear evidence showing an adverse impact on the overall wellbeing of mice imposed upon treatment with 1 mg of m336 antibody before mers-cov challenge (fig. 1) , it is difficult to completely rule out the existence of subtle "yet-to-be investigated" high-dose drug toxicity. we speculate that such a subtle high-dose drug toxicity in the phase of acute and dynamic mers-cov infection initiated at 12 hrs before treatment with 1 mg of m336 could exacerbate drug toxicity, resulting in reduction of appetite and antiviral capacity. however, such a negative impact imposed upon high-dose treatment of virally infected mice appeared to be transient and did not irreversibly alter the final outcome of infection, as judged by the mortality (fig. 2b) . additional studies, especially the pharmacokinetics and the dosing frequency of m336 are warranted in the future to optimize preventive and therapeutic strategies with this promising antibody. the transgenic mice that we used for evaluating the prophylactic and, especially, the therapeutic efficacy of this m336 antibody are extremely sensitive to mers-cov infection and disease, with ld 50 and id 50 of 4.5 and 0.4 tcid 50 of mers-cov, respectively (data not shown), titers which are lower than our original estimations 38 . such a striking ability of this m336 antibody, as a prophylactic or therapeutic agent, to significantly protect these transgenic mice against challenge with 1000 ld 50 of mers-cov is highly impressive. the rbd of the mers-cov, targeted by this m336 antibody, is highly conserved among various clinical isolates and the mutation rate of this rbd appears to be extremely low, compared to that of other rna viruses 23, 28 , thereby making the development of escape mutants to m336 unlikely. however, a combination treatment with multiple neutralizing mabs targeted at different epitopes or the mers-cov-specific hr2p fusion inhibitor targeting the hr1 domain of the s2 subunit of the mers-cov s protein 38,44 could be desirable. by immunizing mice with rbd of mers-cov s protein, li, y. et al. recently developed a humanized mab, named 4c2h, that exhibited strong neutralizing activity with nd 50 of ~0.71 and ~6.25 μ g/ml against the pseudotyped and live mers-cov, respectively 36 , which are about 100-fold less potent than m336 (nd 50 = 0.005 and 0.07 μ g/ml against the pseudotyped and live mers-cov, respectively) 23 . using ad5-hcd26-transduced mouse model 33 , they demonstrated that intravenous administration of a single dose of 4c2h one day before or after the mers-cov challenge resulted in reduction of viral titer by 2 log at 3 dpi. however, intraperitoneal administration of m336 to our hdpp4 tg mice lead to the reduction of viral titer as high as 4 log at 2 dpi. since mers-cov challenged ad5-hcd26-transduced mice showed no severe disease, the effect of 4c2h on the weight loss and mortality in these mice is unavailable. additionally, unlike hdpp4 transgenic mice that we used in this study with well-defined hdpp4 expression as well as 50% lethal dose (ld 50 ) and infectious dose (id 50 ), the intensities of hcd26 expression among the ad5-hcd26-transduecd mice are variable, ranging from undetectable to a high level 45 . although both 4c2h and m336 bind to the rbd of mers-cov s protein, some of the critical amino acid residues recognized by these two mabs are different 29, 36 . the epitope of m336 overlaps extensively with the dpp4-binding site, which is composed of mers-cov rbd residues n501-k502, s504, f506, d510, e513, w535-r542, w553, v555, s557 and s559 29 . the epitope of mab 4c2, the parental mouse mab of 4c2h, only overlaps with partial of dpp4-binding site, which is composed of five rbd residues, w535-e536 and d539-r542. most of other rbd amino acids recognized by 4c2, including y397-n398, k400, l495-k496, p525 and v527-s532, are not located on the dpp4-binding site, indicating that the neutralization efficacy of 4c2h is largely attributed to the steric hindrance created by its binding with mers-cov rbd 36 .these results suggest that combinational use of 4c2h and m336 may exhibit synergistic antiviral effect against both wild-type strains and escape mutants (if any) of mers-cov. taken together, these results suggest to us that the mers-cov rbd protein-specific m336 mab is an excellent candidate for passive immunotherapy to provide immediate and effective protection to individuals who may be exposed to mers-cov and to treat patients who have been exposed. testing in humans is needed for its potential use as a therapeutic for the treatment of mers-cov-infected patients. monoclonal antibody production. for expression of m336 igg1, the previously described m336 igg1 vector 23 was used to infect cho-k1 cells (atcc, manassas, va) with polyfect transfection reagent (qiagen, valencia, ca). after screening of 960 clones for antibody productivity by elisa and subsequent characterization, a stable cell line was generated and inoculated into a bioflo 410 bioreactor (new brunswick scientific, nj) for large-scale production of m336 igg1. purification was carried out by using a protein g column (ge healthcare), and endotoxin was removed by detoxi-gel endotoxin removing columns (thermo scientific) according to the manufacturer's instructions. transgenic mice expressing human dpp4 established by us were used throughout the study. animals were housed in on-site animal facilities at galveston national laboratory under a 12:12 light/dark cycle with room temperature and humidity kept between 21-25 °c and 31-47%, respectively, and with ad libitum access to food and water. all experiments were performed in accordance with the guide of nih and aaalac and were approved by the institutional animal care and use committee at the university of texas medical branch, as described previously 34 . briefly, groups of 6-8-weeks tg mice were challenged intranasally (in) with 10 4 tcid 50 /ml (~1,000 ld 50 ) of mers-cov-emc/2012, originally provided by heinz feldmann (nih, niaid rocky mountain laboratories, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands). the titers of individual virus stocks, stored at − 80 °c, were determined by using vero e6-based infectivity assays and expressed as 50% tissue culture infectious doses (tcid 50 )/ml. scientific reports | 6:31629 | doi: 10.1038/srep31629 viral infections and isolation. all of the animal studies involving infectious mers-cov were conducted within approved animal biosafety level 3 (absl-3) at the galveston national laboratory. experimental designs and strategies in different tg mouse groups involving intranasal challenge with live mers-cov were described in individual experiments in the results section. for live virus isolation, lung tissues were collected at day 2 post mers-cov challenge, weighed, and homogenized in phosphate-buffered saline (pbs) containing 10% fetal calf serum (fcs) by using tissuelyser (qiagen, retsch, haan, germany), as previously described 34 . the resulting suspensions of infected tissues were tittered in the standard vero e6 cell-based infectivity assays to quantify yields of infectious virus expressed as log 10 tcid 50 per gram (g) of tissue. rna extraction and viral titers determination by real-time q-pcr. lung tissue samples from each group of mice were transferred to individual vials having rna later solution (qiagen) and subsequently homogenized and subjected to total rna isolation, by using trizol reagent (life technologies), to assess mers-cov-specific genome targeting of virus-specific upstream e gene (upe) and endogenous control gene (mouse β -actin) by using a one-step rt-pcr kit (invitrogen), as previously described 34 . ct values for each sample were analyzed against ct values generated in our lab from the standard curve of mers-cov mrna copy number. relative mers-cov upe mrna expression value was calculated for each replicate and expressed as the equivalent of log10 tcid 50 per gram (g) of the tissue by the standard threshold cycle (∆∆ct) method. ct value analysis was done by using bio-rad cfx manager 3.0 software. is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sarslike pandemic? hospital outbreak of middle east respiratory syndrome coronavirus middle east 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prophylaxis with a mers-cov-specific human monoclonal antibody protects rabbits from mers-cov infection rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the growth and potential of human antiviral monoclonal antibody therapeutics the spike protein of sars-cov-a target for vaccine and therapeutic development passive immunity in prevention and treatment of infectious diseases prophylaxis and therapy for chikungunya virus infection post-exposure treatment of ebola virus using passive immunotherapy: proposal for a new strategy structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor we thank dr. heinz feldmann, national institute of health at hamilton, montana, and dr. ron a. fouchier, erasmus medical center at rotterdam, the netherlands for the mers-cov. this research was supported in part by a national institutes of health grant, r21ai113206-01 (to c-t.k.t), and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (grant number: 5uc7ai094660-05. project title: national biocontainment laboratories (nbls) operations support), university of texas medical branch, galveston, tx (to c-t.k.t.), intramural funding of nci (to dsd), and the national natural science foundation of china (#31570936 to sj, #31570936 to ty). t.g was supported in part by a t32 biodefense training program (5 t32 ai 60549-12) awarded to utmb by nih. histopathology. mice were necropsied, lung tissues were inflated and fixed in 10% neutral buffered formalin for 3 days before paraffin-embedded and processed for routine hematoxylin and eosin stain (h&e) for assessing the histopathology 46 . key: cord-332952-d5l60cgc authors: nan title: mers: progress on the global response, remaining challenges and the way forward date: 2018-09-17 journal: antiviral res doi: 10.1016/j.antiviral.2018.09.002 sha: doc_id: 332952 cord_uid: d5l60cgc this article summarizes progress in research on middle east respiratory syndrome (mers) since a fao-oie-who global technical meeting held at who headquarters in geneva on 25–27 september 2017. the meeting reviewed the latest scientific findings and identified and prioritized the global activities necessary to prevent, manage and control the disease. critical needs for research and technical guidance identified during the meeting have been used to update the who r&d mers-cov roadmap for diagnostics, therapeutics and vaccines and a broader public health research agenda. since the 2017 meeting, progress has been made on several key actions in animal populations, at the animal/human interface and in human populations. this report also summarizes the latest scientific studies on mers since 2017, including data from more than 50 research studies examining the presence of mers-cov infection in dromedary camels. since its identification in the kingdom of saudi arabia (ksa) (zaki et al., 2012) and jordan (hijawi et al., 2013) in 2012, middle east respiratory syndrome (mers) has become a global public health threat. typical of an emerging zoonosis, middle east respiratory syndrome coronavirus (mers-cov) has an animal reservoir, i.e. dromedary camels in which the virus causes little to no disease (mohd et al., 2016) . many details about the extent of circulation and the mechanisms of transmission within dromedary camel herds, or factors related to zoonotic transmission and differences in circulating mers-cov strains, remain unknown. the virus has repeatedly spilled over from dromedary camels to humans, principally in countries on the arabian peninsula, causing significant morbidity and mortality (world health organization, 2017a; azhar et al., 2014) . clusters of cases in the community and among family members are rare (world health organization, 2017a; . however, delays in diagnosis in hospitals has sometimes led to secondary cases among health care workers, patients sharing rooms or family members as a result of unprotected direct contact with a patient before isolation. this humanto-human transmission in health care facilities can sometimes be amplified, causing very large outbreaks, as has been seen in the middle east and in the republic of korea, with significant public health and economic impacts (hijawi et al., 2013; assiri et al., 2013; drosten et al., 2015; al hosani et al., 2016; ki, 2015; park et al., 2015) . as of august 2018, more than 2249 human cases from 27 countries have been reported to the world health organization (who) (world health organization, 2017a) . the fao, oie and who tripartite have regularly brought together affected member states, public health and animal officials, and academics to discuss what is known and unknown about the zoonotic origin of mers-cov (world health organization, 2016; fao, 2016 fao, , 2014 ; who regional office for the eastern mediterranean, 2013a). the purposes of these meetings and workshops have been to advocate for more surveillance and research on mers-cov in animals and humans, to share information about how mers-cov is transmitted between animals, from animals to humans and between humans, to describe the diseases it causes, and to develop policies and guidelines for detection, https://doi.org/10.1016/j.antiviral.2018.09.002 received 30 august 2018; accepted 4 september 2018 reporting of animal and human infections, and prevention of human cases and clusters. in the two years since the last international technical consultation on mers-cov in 2016 13 , there have been notable improvements in surveillance and reporting of human cases, multidisciplinary research, cross-sectoral collaboration at country level, public awareness about the disease, and laboratory and surveillance capacity in affected countries. in addition, a number of countries in the arabian peninsula and in africa have engaged in research activities and surveillance of camel populations to shed light on the wider distribution of this virus or investigate transmission patterns and routes for viral shedding. as a follow-up to previous meetings (world health organization, 2016; fao, 2016 fao, , 2014 ; who regional office for the eastern mediterranean, 2014; who regional office for the eastern mediterranean, 2013b, 2013c), fao, oie and who tripartite held a global technical meeting on mers-cov with representatives from ministries of health and ministries of agriculture, subject matter experts, researchers, funders and industrial partners from 25 to 27 september 2017 in geneva, switzerland (see supplementary information) (world health organization et al., 2017) . the objectives were to review the latest scientific evidence on mers-cov, further enhance cross-sectoral collaboration and communication during preparedness and response activities, and identify research priorities given the advancements in our knowledge. with 130 participants, this was the largest mers-cov technical meeting to date and the first meeting attended by representatives from both affected and at risk countries. that is, countries which have reported human infection, countries with evidence of mers-cov in dromedary camels but no reported human cases, and countries at risk for importation (countries without infected camels that have close ties to affected countries through expatriate workers, travel to affected countries for medical procedures and/or frequent international travel). there is strong consensus among all stakeholders that dromedary camels are the main source of transmission to humans. in 2014, oie identified mers-cov as an emerging disease with zoonotic potential in camels and thereby creating expectations of reporting positive camels by countries (oie, 2014a) and recently published a mers-cov case definition (oie, 2017) for the reporting of confirmed and suspected infection in camels. not all countries face the same risks. for example, countries that have the infected reservoir (dromedary camels) differ from those countries in which dromedary camels show no evidence of current or past infection (fig. 1 ). there may also be differences in spillover potential in countries with documented zoonotic transmission, compared to those without, due to several factors including potential differences in husbandry practices, cultural, social, medicinal, occupational exposures, prevalence of underlying chronic medical conditions, or genetic factors in human populations, and mers-cov viral differences (wong et al., 2015) . as such, technical and risk mitigation guidance to protect human health and research priorities differ by region. the findings from the global technical meeting are summarized below: i. surveillance needs: surveillance in animals and humans to limit zoonotic transmission routine human surveillance for mers-cov in ksa (abdulaziz et al., 2017) and throughout the middle east has improved since the identification of the virus in humans in 2012, but there is significant variation in the quality and extent of surveillance between countries. in other parts of the world, surveillance is limited. since it is known that mers-cov is enzootic in areas of africa and asia where dromedary camels are found, heighted awareness and surveillance for zoonotic mers is required. this is currently lacking and remains a knowledge gap. one exception is the notable effort to identify potential mers-cov infection among pilgrims travelling back from the middle east. since 2012, event-based surveillance among pilgrims returning from hajj, umrah and other religious events in ksa has been conducted by ksa and countries sending pilgrims. while many return reporting respiratory symptoms, no mers-cov infections have been identified among returning pilgrims (muraduzzaman et al., 2018; barasheed et al., 2014; atabani et al., 2016; koul et al., 2017; annan et al., 2015; ma et al., 2017; memish et al., 2014a memish et al., , 2014b refaey et al., 2017; al-abdallat et al., 2017; matthew et al., 2015; alqahtani et al., 2016; win et al., 2016; yavarian et al., 2018; kapoor et al., 2014) . among animals, field surveys conducted to date have included several domestic and wildlife species including dromedary camels (camelus dromedarius) and bactrian camels (camelus bactrianus), goats, bats, cattle, sheep, chickens, swine, ducks, buffalo and equids. field studies in dromedary camels have been conducted in a number of countries (table 1) . to date, mers-cov rna or mers-cov-specific antibodies have been identified in dromedary camels a number of countries (table 1 ) except australia (hemida et al., 2014) , kazakhstan (miguel et al., 2016) , and the netherlands (reusken et al., 2013a) . other livestock such as alpacas (vicugna pacos), llamas (llama pacos), young goats, rabbits and pigs have been shown to be susceptible to experimental infection (crameri et al., 2016; adney et al., 2016; vergara-alert et al., 2017) . despite improvements, routine surveillance in dromedary populations is limited. the lack of surveillance information about mers-cov circulation in dromedary camels restricts our understanding of the transmission dynamics and epidemiology in dromedary camel populations. meeting participants agreed that surveillance should be integrated into existing surveillance systems, particularly in at-risk countries, similar to one health approaches developed for avian influenza, and existing human respiratory disease surveillance systems set up for influenza-like illness (ili) or severe acute respiratory infections (sari). currently, a limitation in our ability to mitigate spillover from dromedary camels to humans is a lack of clarity on the mode(s) of transmission between dromedary camels and humans, the extent and epidemiology of mers-cov circulation in dromedary camels in large parts of africa and south asia, and on why zoonotic transmission is limited across africa, large parts of the middle east, and some parts of south asia despite high seroprevalence in dromedary camels (chu et al., 2018) (table 1) . fao has outlined the meeting participants conclusions on priorities for mers-cov surveillance and management of pcr positive dromedary camels, coordinated outbreak investigation of community acquired cases with dromedary exposure, testing of animals at quarantine and entry points, food safety and environmental contamination, risk communication and awareness raising for mers-cov among animal owners and intersectoral collaboration and coordination in an updated doha declaration first published in 2015 (fao, 2015) (ref/hyperlink). in dromedary camels, longitudinal studies to evaluate the natural history, shedding profile and immunity were highlighted as key research priorities. meeting participants agreed that further understanding of differences in viral strains and transmission dynamics, including the role of immunity in acquiring infection and shedding virus, the geographic range of spillover events, and environmental, behavioral or host-related risk factors for zoonotic transmission should be prioritized. countries face significant challenges in the early identification and diagnosis of mers in humans due to the non-specificity of clinical symptoms ( the spectrum of illness ranges from no symptoms (or asymptomatic infection) to severe disease including pneumonia, acute respiratory disease syndrome, organ failure and death, with a case fatality ratio 35.5% among reported cases (world health organization, 2012). the delay in identification and recognition of signs and symptoms compatible with mers and delay in early isolation of patients has reduced the ability to prevent transmission between people in health care settings, notably in emergency departments, cardiac care centers and renal dialysis units (hijawi et al., 2013; assiri et al., 2013; drosten et al., 2015; al hosani et al., 2016; ki, 2015; park et al., 2015; ahmed et al., 2018; amer et al., 2018) . our understanding of human-to-human transmission in health care settings has improved through experimental and observational studies conducted in countries during such outbreaks. for example, studies of respiratory pathogens (yu et al., 2007; tran et al., 2012; thompson et al., 2013) and mers-cov conducted in the middle east (assiri et al., 2013; oboho et al., 2015; hunter et al., 2016; balkhy et al., 2016) and the republic of korea (bin et al., 2016; kim et al., 2016a kim et al., , 2016b nam et al., 2017) illustrate that aerosol-generating procedures and non-invasive ventilation, combined with inappropriate infection prevention and control practices and lack of adherence to standard practices had an important role in facilitating human-to-human transmission in health care settings. the role of environmental contamination has been evaluated in a number of hospitals following the 2015 outbreak in the republic of korea and collaborative, experimental studies are being conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air (bin et al., 2016; kim et al., 2016a; van doremalen et al., 2013) . the role of mild or asymptomatic cases in transmission chains, however, remains unclear (omrani et al., 2013; memish et al., 2014c; al-gethamy et al., 2015; moon and son, 2017; al-abdely et al., 2018) , warranting targeted epidemiological and clinical studies to be conducted among contacts during outbreaks, especially in health care facilities. countries which have reported health care-associated outbreaks have implemented a variety of strategies to improve infection prevention and control and reduce human-to-human transmission in hospitals, including the introduction of a visual triage system prior to entrance to the emergency departments, the restructure of emergency department layouts for better triage of patients with respiratory symptoms, the standardization and training and re-training of infection prevention and control practices at facilities with high hospital staff turnover, and the auditing of health care facilities for adherence to infection prevention and control measures. iii. product research and development needs: clinical management, diagnostics and medical interventions the who r&d blueprint, a global strategy and preparedness plan that allows the rapid activation of r&d of epidemic pathogens, aims to fast-track the development and use of effective point-of-care diagnostic tests, vaccines and medicines that can be used to save lives and avert large scale crises. since 2015, mers-cov has been included in the annual who r&d blueprint list of prioritized pathogens for accelerated research and development on diagnostics, vaccines and therapeutics (world health organization). in addition, mers-cov has a specific roadmap for product research and development, outlined by who in ( the nonspecific, and sometimes unusual, clinical presentation of mers in humans, makes early diagnosis difficult in health care facilities. while several highly specific and sensitive molecular and serologic assays exist for diagnosis in animals and humans corman et al., 2012a corman et al., , 2012b lu et al., 2014; perera et al., 2013a; reusken et al., 2013b; müller et al., 2015; song et al., 2015) , there was a clear call from representatives from affected countries for the development of a rapid diagnostic test to improve identification and isolation of primary human cases in health care facilities. a full landscape analysis of mers-cov diagnostics will be published separately (van kerkhove, personal communication). at the global technical meeting, several therapeutics (including convalescent plasma, lopinavir/ritonavir, ribavirin, interferon and novel therapies including polyclonal antibodies and broad-spectrum antivirals) in development were presented. however, small case numbers make the evaluation of their impact on morbidity and mortality from mers-cov infection difficult (arabi et al., 2017a; arabi et al., 2017b; al-dorzi et al., 2016; sheahan et al., 2017; ko et al., 2018; arabi et al., 2017c) . several pre-clinical and phase i-iii studies are under way or in the design phase (outlined by the who r&d blueprint: http://who-blueprint-mapping-tool.surge.sh/). who is currently evaluating all available evidence on therapeutics to update guidance on clinical management of patients and in the process to develop standardized clinical trial protocols that could be used in affected countries to evaluate promising therapeutic candidates. who has identified target product profiles for mers-cov vaccines which include a dromedary camel vaccine for the reduction of zoonotic transmission, a human vaccine for long term protection of high risk individuals, such as those working with infected dromedary camels or health care workers, and a human vaccine for reactive use in outbreak settings (world health organization, 2017b) . currently, no mers-cov-specific or licensed human vaccines are available (modjarrad et al., 2016; world health organization, 2017c) . several human vaccine candidates for coronaviruses, including mers-cov, are at various stages of development and five general vaccine technology platforms have been developed and target the mers-cov spike protein (modjarrad et al., 2016; okba et al., 2017) . who, the ministry of health in ksa and the international vaccine institute (ivi) have continued to further align efforts to develop coronavirus vaccines (excler et al., 2016) and the coalition for epidemic preparedness and innovation (cepi) has included mers-cov as one of three priority pathogens for financing of a human vaccine. understanding correlates for protection and having a reliable animal model remain essential for evaluating coronavirus vaccine candidates, including mers-cov. there was a clear call from global technical meeting participants to accelerate the development of a dromedary camel vaccine in order to evaluate the potential to reduce spillover transmission to humans. the acceptability, cost-effectiveness and feasibility of a dromedary camel vaccine will also need to be evaluated and compared to other intervention strategies, such as human vaccination of high risk groups (e.g., those with occupational exposure). because mers-cov is endemic in dromedary camel populations in the middle east and elsewhere, multiple intervention strategies, including personal protective measures and the strategic implementation of a camel vaccine, are likely needed to reduce transmission from dromedary camels to humans. (farag et al., 2015) antibodies to mers-cov s1 were found in 100 of 103 (97.1%) dromedary camels tested by micro-array technology (farag et table 2 list of prioritized research and progress on mers-cov research, as discussed at the september 2017 meeting. *based on an enhanced understanding of the virus, the doha declaration (fao, 2015) is undergoing revision with a focus on guiding surveillance techniques, management of dromedary camels shedding the virus, research, regional and inter-sectoral coordination, risk communication, food and environmental safety practices, and biosecurity measures. the update includes explicit guidance on import testing, quarantine procedures, and management of shedding animals. these recommendations and priority actions in the doha declaration will be delivered as a separate document after validation by stakeholders in affected and at risk countries. at least two promising camel vaccine candidates are currently in development and being evaluated in field trials (haagmans et al., 2016; alharbi et al., 2017) . stakeholders agreed that the current funding mechanisms need to include risk-mitigating options that target the animal-human interface to prevent zoonotic transmission. these funding pathways would enable the development of camel vaccine candidates. stakeholders also agreed that prior to camel vaccine implementation, consultation with camel owners and government agencies, feasibility studies including exploring opportunities for commercial manufacturing and incentives for camel vaccination and an assessment of potential trade implications, would all be critical. in june 2018, who and the international vaccine institut (ivi) held a joint workshop update the status of human and animal mers-cov vaccine development, and identify and prioritize activities to accelerate vaccine research and development. the meeting was held in seoul, korea on 26-27 june and included 120 experts and professionals from industry, academia, international organizations and government agencies around the world, including the coalition for epidemic preparedness innovations (cepi), the korean ministry of food and drug safety (kmfds) and the korea centers for disease control and prevention (kcdc). the global technical meeting served as an opportunity to review the available evidence and best practices for control of this epidemic threat six years after the virus was first detected in humans. this covered our understanding of the virus, our ability to detect and respond to cases in animal and human populations, how we communicate our findings and how our work impacts policy decisions to protect animals and prevent human infections. during the global technical meeting, the latest findings from scientific studies and knowledge gained from collaborative research and surveillance were shared across animal, environmental and human sectors. fao, oie and who strongly believe that to effectively address zoonoses, including mers-cov, a one health approach to prevent, detect, contain, eliminate and respond to animal and public health risks from zoonotic high threat respiratory pathogens such as mers-cov and should involve all relevant sectors, the public and animal health and academic research community, industry and affected communities. we acknowledged the progress that has been made, and importantly, discussed the challenges that need to be addressed so that we can minimize the future public health and economic impacts of this epidemic prone virus. our aim was to articulate a clear action plan to address these remaining unknowns and to foster better collaboration between sectors and with subject matter experts willing to support member states. given the marked expansion in research related to mers-cov conducted in the past two years, fao, oie and who agree that global surveillance and research activities must now be focused on achieving the following major public health goals: reducing zoonotic transmission, detecting and identifying suspected cases early, providing safe and effective treatment to reduce human morbidity and mortality, and significantly reducing human-to-human transmission in health care settings. the critical needs for research and technical guidance identified during the global technical meeting have been used to inform the who r&d mers-cov roadmap (modjarrad et al., 2016 ) and a broader research agenda for mers-cov and other high threat coronaviruses. this research agenda serves as a catalyst to focus, align and mobilize partners to address outstanding knowledge gaps in relation to mers-cov across five technical areas: i) virus origin and characteristics, ii) epidemiology and transmission, iii) clinical management and infection prevention and control, iv) product development and implementation (modjarrad et al., 2016) and v) impact of interventions and operational research. the meeting identified a large number of remaining priorities and the organizing committee has summarized the 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isolation of a novel coronavirus from a man with pneumonia in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia we gratefully acknowledge the input from all those who attended the fao-oie-who global technical meeting on mers-cov in september 2017. the authors also thank malik peiris for his review of the final manuscript. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. the authors have no competing financial interests. supplementary data to this article can be found online at https:// doi.org/mmcdoino. key: cord-306004-amv0los1 authors: widagdo, w.; sooksawasdi na ayudhya, syriam; hundie, gadissa b.; haagmans, bart l. title: host determinants of mers-cov transmission and pathogenesis date: 2019-03-19 journal: viruses doi: 10.3390/v11030280 sha: doc_id: 306004 cord_uid: amv0los1 middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. in dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in mers-cov pathogenesis and transmission. one of these host factors, the mers-cov receptor dipeptidyl peptidase-4 (dpp4), may be a critical determinant because it is variably expressed in mers-cov-susceptible species as well as in humans. this could partially explain interand intraspecies differences in the tropism, pathogenesis, and transmissibility of mers-cov. in this review, we explore the role of dpp4 and other host factors in mers-cov transmission and pathogenesis—such as sialic acids, host proteases, and interferons. further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that was isolated in late 2012 [1] . since then, the virus has caused multiple outbreaks and infected more than 2000 individuals, [2] who then develop a respiratory infection ranging in severity from asymptomatic to fatal [3, 4] . severe-to-fatal mers-cov patients have a higher chance of transmitting this virus since they shed a higher amount of virus progeny in comparison to the asymptomatic-to-mild ones [5] [6] [7] [8] . identifying and quarantining these patients in healthcare facilities where outbreaks have occurred, together with implementing proper infection control, has been effective in reducing transmission and containing these outbreaks [9, 10] . however, new mers-cov cases are still being reported, especially in the arabian peninsula [2, 11] . this is partly due to the continuous zoonotic introduction of this virus to the human population in this region by dromedaries [12] . the dromedary camel is the only animal species that has been reported to transmit this virus to humans [13] [14] [15] [16] . mers-cov infection in these animals merely causes mild upper respiratory tract infection [17, 18] , but seroepidemiological studies showed that this virus has been circulating in dromedary camels for decades, suggesting the efficient transmission of mers-cov in this species [19] [20] [21] [22] . although the clinical manifestations, as well as transmission, are remarkably different in mers-cov-infected humans and dromedary camels, the viruses isolated from these two species are highly similar, if not indistinguishable [12, 16] . this indicates that host factors play a significant role in mers-cov pathogenesis and transmission. however, the identity of these host factors and how they affect the pathogenesis and transmission of mers-cov are generally not well understood. dipeptidyl peptidase-4 (dpp4)-the mers-cov receptor, sialic acids, proteases, and interferons are ; a cartoon representation of mers-cov s1 protein binding to dpp4 (pdb code 4l72) (b). the s protein consists of the s1 and s2 subunits. the α/β hydrolase domain of dpp4 is indicated in red, β-propeller domain in green, while part of the mers-cov s1 protein is shown in blue. other mers-cov-interacting host factors besides dpp4 are less extensively studied and have mostly been investigated in vitro. glycotopes of α2,3-sialic acids coupled with 5-n-acetylated neuraminic acid are recognized by the s1 protein of mers-cov during attachment [24] . in the absence of these glycotopes, mers-cov entry is reduced but not abolished, indicating their function as an attachment factor rather than a receptor [24] . besides α2,3-sialic acids, ceacam5 and grp78 have also been suggested to be attachment factors for mers-cov, but their roles in vivo during mers-cov infection are not clear at this moment [37, 38] . post attachment, mers-cov uses the c-terminal part of its s protein-known as s2 ( figure 1a )-to interact with host proteases, such as furin, tmprss2, and cathepsins [39] [40] [41] [42] . these proteases cleave the s protein and induce conformational changes, allowing fusion between viral and host cellular membranes, resulting in the release of viral rna into the cell cytoplasm [27] . tmprss2 and dpp4 are held in one complex at the cell surface by a scaffolding protein, the tetraspanin cd9, leading to a rapid and efficient entry of mers-cov into the susceptible cells [43] . once fusion with host cell membranes has occurred, mers-cov subsequently replicates its genetic material and produces viral proteins in the cell cytoplasm to generate new virus progeny. during this stage, mers-cov uses its nsp3-4 polyproteins to build its replication organelles as well as its accessory proteins such as the 4a and 4b proteins to inhibit host anti-viral defense mechanisms [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] . however, the capacity of mers-cov accessory proteins to impede several pathways of host immune response in the lungs may be limited. mers-cov inoculation of macaques and genetically modified mice generally results in limited clinical manifestations; thus, adapting this virus through serial passaging or defecting the type i interferon pathway may be needed to enhance viral replication and pathogenesis in these animals [32, [55] [56] [57] [58] . these observations, together with studies showing type i interferon capacity to inhibit mers-cov infection in vitro [25, 59] , highlight the importance of the innate immune response, especially type i interferon, as an inhibiting factor for mers-cov. so far mers-cov has been isolated from dromedary camels and humans [1, 60] . both species are not only susceptible to mers-cov infection, but also capable of transmitting this virus [7, [12] [13] [14] [15] [16] [17] [18] 22 ]. however, current data indicate that virus spread is more efficient in dromedary camels than in humans [5, 7, [19] [20] [21] 61] . this difference in transmissibility could be partially due to the different tropism of mers-cov in these two species. in dromedaries, mers-cov has been shown to replicate in the nasal epithelium upon experimental in vivo infection [17] , while in humans, mers-cov mainly replicates in the lower respiratory tract, particularly in the bronchiolar and alveolar epithelia [23, [62] [63] [64] [65] . higher viral rna levels in the sputum and lavage samples of mers-cov patients compared to nasal and throat swabs are consistent with the tropism of mers-cov in humans [66] [67] [68] . this different mers-cov tropism in dromedary camels and humans is in line with the localization of dpp4 in the respiratory tract tissues of these two species. in humans, dpp4 is absent in the nasal epithelium but present in the lower respiratory tract epithelium, mainly in type ii pneumocytes [69, 70] . in contrast, dpp4 is expressed in the nasal epithelium of dromedary camels [69] . this difference in dpp4 localization between humans and dromedary camels therefore explains mers-cov tropism in these two species and highlights dpp4 as an essential determinant of mers-cov tropism. dpp4 localization has also been investigated in many other mers-cov-susceptible species. in gambian and egyptian fruit bats, dpp4 is expressed in the respiratory tract and intestinal epithelium, suggesting that mers-cov can target both tissues [71] . in line with this finding, mers-cov inoculation via intranasal and intraperitoneal routes in the jamaican fruit bat led to viral rna shedding both in the respiratory tract and the intestinal tract [72] . in contrast to frugivorous bats, dpp4 is limitedly expressed in the respiratory tract epithelium of two insectivorous bats, i.e., common pipistrelle and common serotine bats, but abundant in their intestinal epithelium [71] . accordingly, sequences of mers-like-covs were mainly obtained from rectal swabs and fecal samples of insectivorous bats [73] [74] [75] [76] [77] [78] [79] [80] . these findings not only support insectivorous bats as the origin host of mers-cov [73] [74] [75] [76] [77] [78] [79] [80] , but also indicate the importance of intestinal tropism and fecal-oral transmission of mers-like-cov in these insectivorous bats. besides bats, humans, and dromedary camels, other animal species have also been proposed as potential hosts of mers-cov. remarkably, dpp4 of horses, llamas, alpacas, pigs, bovines, goats, sheep, and rabbits has been demonstrated to recognize the s protein of mers-cov [81, 82] . in most of these species, there is a preferential upper respiratory tract expression of dpp4 observed. rabbits express dpp4 in the upper and lower respiratory tract epithelium, and thus may allow mers-cov to replicate in both compartments [33, 83] . horses, llamas, and pigs mainly express dpp4 in the upper respiratory tract-particularly the nasal epithelium [84] . upon intranasal mers-cov inoculation, llamas, alpacas, and pigs developed upper respiratory tract infection, while horses did not seroconvert and only shed infectious virus in a limited amount [84] [85] [86] [87] [88] . the reason why horses seem to be less permissive to mers-cov remains to be investigated, but a chronic co-infection in the guttural pouch, a common disease among horses, might be one of the explanations. this guttural pouch infection results in excessive mucus production that might hinder mers-cov from attaching and entering the nasal epithelium [84, 89, 90] . sheep, on the other hand, did not seem to express significant levels of dpp4 in their respiratory tract, and thus did not seroconvert nor shed infectious virus upon experimental mers-cov inoculation [84, 88] . comparable to sheep, goats limitedly shed infectious virus upon experimental infection and did not transmit this virus to other naïve goats upon direct contact [88] . the results of experimental mers-cov infection in livestock animals are in line with data from epidemiological studies. mers-cov seropositive llamas and alpacas are present in the field, while horses, goats, and sheep are generally found to be seronegative [22, 86, 87, [91] [92] [93] [94] [95] [96] [97] [98] . given the fact that experimental in vivo infection studies and dpp4 expression analysis in different animal species revealed that dromedary camels are not the only animals in which mers-cov has an upper respiratory tract tropism [17, 18, 83, 84] , it is then relevant to question whether other animals can potentially spread mers-cov as well. new world camelids, i.e., alpacas and llamas, are able to transmit the virus to respective naïve animals upon contact [86] . pigs and rabbits, on the other hand, hardly transmit the virus-neither by contact nor airborne routes [83, 99] . most likely, this is caused by the fact that pigs and rabbits, unlike dromedary camels, shed low levels of infectious virus upon mers-cov inoculation ( figure 2) . this difference indicates that other host factors besides dpp4 could cause interspecies variation in mers-cov infection. indeed, several glycotopes of α2,3-sialic acids that function as attachment factors of mers-cov are present in the nasal epithelium of dromedary camels but absent in that of rabbits and pigs ( figure 3 ) [24, 100] . the lack of these glycotopes in pigs and rabbits might limit the susceptibility and transmission of mers-cov in these animals. although the role of these glycotopes in mers-cov transmission still requires further investigation, it remains plausible that an efficient transmission of this virus might require the presence of both dpp4 and mers-cov-recognized glycotopes of α2,3-sialic acids (figure 3 ). . it has also been reported that the lysosomal proteases from bat cells support coronavirus spike-mediated virus entry more efficiently than their counterparts from human cells [39] . these observations suggest that host proteases from different host species may determine the species and tissue tropism of mers-cov. because mers-cov has been circulating in dromedary camels for decades before emerging in the human population [19] [20] [21] [22] , it is plausible that this virus inhibits the immune response of dromedary camels more efficiently than that of other species, including pigs and rabbits. the difference in immune response among mers-cov-susceptible species is therefore another factor that might yield interspecies variation in permissiveness to mers-cov. characterizing the difference in host proteases and immune responses among mers-cov-susceptible species, as performed for dpp4 and mers-cov-recognized α2,3-sialic acid glycotopes (figure 3) , has not yet been investigated. these data, however, may further explain interspecies variation in mers-cov infection and transmission. mers-cov causes respiratory infection in humans ranging from asymptomatic to severe pneumonia [3, 4] . however, it is currently unclear what causes this intraspecies variation. epidemiology data indicate that individuals with certain risk factors are at higher risk of developing severe mers-cov infection [4, 102] . this implies that some host factors may dictate the outcome of mers-cov infection, thus rendering intraspecies variation. two of the risk factors, i.e., smoking and chronic obstructive pulmonary disease (copd), have been shown to upregulate dpp4 expression in the lungs [70, [102] [103] [104] , suggesting dpp4 as a possible reason for intraspecies variation observed among mers-cov patients. in healthy human lungs, dpp4 is almost exclusively expressed in type ii pneumocytes [69, 70] . type ii pneumocytes are small cuboidal cells that can regenerate alveolar epithelium upon injury, and roughly cover 2% of the alveolar surface area. meanwhile, around 95% of the surface area of the alveolus is occupied by type i pneumocytes that are morphologically flat and responsible for gas exchange [105, 106] . in the lungs of smokers and copd patients, unlike in healthy human lungs, dpp4 is prominently expressed in both type i and ii pneumocytes, indicating upregulated expression on type i pneumocytes [104] . autopsy reports from fatal mers-cov patients showed that both type i and ii pneumocytes expressed dpp4 and became infected by mers-cov, proposing a role of dpp4-expressing type i pneumocytes in mers-cov pathogenesis [64, 107] . damage to type i cells in the lung alveoli during viral infection may lead to diffuse alveolar damage [108] . in line with observations made in human mers cases, common marmosets that express dpp4 in both type i and ii pneumocytes have been reported to produce more infectious virus upon experimental mers-cov infection, compared to rhesus and cynomolgus macaques that merely expressed dpp4 in type ii pneumocytes [58, [109] [110] [111] [112] . accordingly, these common marmosets developed moderate-to-severe infection, while macaques generally developed mild transient pneumonia [32, 58, [109] [110] [111] [112] . similarly, in genetically modified mice that displayed mers-cov tropism for type ii pneumocytes, only mild clinical manifestations were observed upon mers-cov infection [56, 113] . adapting mers-cov through serial passaging or upregulating dpp4 expression throughout the airway epithelium in mice, however, will induce severe clinical disease [55, 56] . these data altogether support the role of dpp4-expressing type i pneumocytes in the pathogenesis of severe mers-cov infection. the differential expression of host factors that limits the infection should also be taken into account. dpp4 in soluble form has been demonstrated to protect against mers-cov infection in vitro and in a mouse model [23, 114] ; however, its presence in the lungs and role in mers-cov pathogenesis remain to be investigated. the host immune response also has the capacity to inhibit mers-cov infection. mers-cov has been shown to replicate to higher levels in immunocompromised rhesus macaques [115] , consistent with the observation that immunocompromised individuals have difficulties clearing mers-cov upon infection [68, 107, 116] . the survivors of mers-cov infection have been shown to develop virus-specific cd4+ and cd8+ t cell responses, implying the role of t cells in virus clearance [117] . however, the depletion of t cells in mice can either lead to failure in mers-cov clearance or improvement in clinical outcome, depending on the type of mouse model used [57, 118] . therefore, the role of adaptive immune response in mers-cov pathogenesis is currently unclear. on the other hand, one of the main components of the host innate immune response, type i interferon, inhibits mers-cov replication in susceptible cells, partly by inhibiting double membrane vesicles (dmv) formation [25, 57, 59, 119, 120] . the absence of type i interferon signaling in mice also resulted in more severe clinical manifestations and histopathological lesions upon mers-cov infection [57] . advance age, which can cause delayed type i interferon response upon viral infection, is a well-known risk factor for fatal mers-cov infection [4, 102, [121] [122] [123] . collectively, these data highlight the role of host innate immune response as a potent inhibitor for mers-cov infection. it is indubitable that severe mers-cov infection is not solely driven by the pathogen. additional underlying conditions increase mers-cov replication and induce severe-to-fatal clinical manifestations [4, 11, 103, 124, 125] . it is plausible that more than one underlying condition is needed to yield a fatal outcome. dpp4 upregulation in type i pneumocytes and insufficient type i interferon response might be crucial determinants for severe mers-cov infection (figure 4 ). further investigation of the host determinants of mers-cov pathogenesis may offer insights for developing novel therapeutic measures. although mers-cov has been reported to undergo some genotypic changes since it emerged in the human population [12, [126] [127] [128] [129] , this has not resulted in distinct phenotypic changes so far [63, 126] . therefore, host factors remain the most significant determinant in explaining inter-and intraspecies variations observed in mers-cov pathogenesis and transmission. dpp4 and mers-cov-recognized α2,3-sialic acids might partially explain these variations, since their localization has been demonstrated to be variable between mers-cov-susceptible species [69, 71, 84, 100] . dpp4 expression in human lungs has also been shown to vary due to certain comorbidities [70, 96, 104] . nevertheless, it is undoubtable that the inter-and intraspecies variation in mers-cov pathogenesis and transmission is a complex phenomenon influenced by more than one host factor. current data suggest proteases and interferons as other critical host factors, but how they instigate inter-and intraspecies variations, as well as their role in mers-cov pathogenesis and transmission, still remain to be further elucidated. 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cord-265380-2gs34xcw authors: leist, sarah r.; cockrell, adam s. title: genetically engineering a susceptible mouse model for mers-cov-induced acute respiratory distress syndrome date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_12 sha: doc_id: 265380 cord_uid: 2gs34xcw since 2012, monthly cases of middle east respiratory syndrome coronavirus (mers-cov) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. the ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with mers-cov in human infections. the inability of mers-cov to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. identification of human dipeptidyl peptidase iv (hdpp4) as the receptor for mers-cov infection opened the door for genetic engineering of mice. precise molecular engineering of mouse dpp4 (mdpp4) with clustered regularly interspaced short palindromic repeats (crispr)/cas9 technology maintained inherent expression profiles, and limited mers-cov susceptibility to tissues that naturally express mdpp4, notably the lower respiratory tract wherein mers-cov elicits severe pulmonary pathology. here, we describe the generation of the 288–330(+/+) mers-cov mouse model in which mice were made susceptible to mers-cov by modifying two amino acids on mdpp4 (a288 and t330), and the use of adaptive evolution to generate novel mers-cov isolates that cause fatal respiratory disease. the 288–330(+/+) mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for mers-cov. the chapter starts with a historical perspective on the emergence of mers-cov and animal models evaluated for mers-cov pathogenesis, and then outlines the development of the 288–330(+/+) mouse model, assays for assessing a mers-cov pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice. in february of 2018 mers-cov was listed as a priority on the r&d blueprint for the global strategy and preparedness plan outlined by the world health organization (who) [1] . the r&d blueprint includes viruses that pose a global public health risk, and for which there are no available therapeutic countermeasures [1] . twenty-seven countries have reported cases of mers-cov with most cases confined to the arabian peninsula. diagnosed cases of mers-cov in countries outside the arabian peninsula are primarily traveler associated. the potential for global spread of mers-cov was realized in 2015 when a single traveler returning to south korea initiated an outbreak that infected 186 people resulting in 20% fatality and caused widespread fear that crippled the economy for nearly 6 months [2] [3] [4] . human-to-human transmission is often associated with close contact in the health care setting, but can also occur between family members within a household [5] . asymptomatic individuals pose a particular risk of transmission due to their unknown carrier status as demonstrated in the health care setting [6] . despite the high percent of fatalities associated with mers-cov outbreaks on the arabian peninsula most epidemiological studies suggest r 0 values <1, indicative of a low risk of sustainable human-to-human transmission, whereas epidemiological studies from the south korean outbreak describe r 0 values (>1) akin to more sustainable human-to-human transmission [7] . recurring spillover events from dromedary camels (zoonotic reservoir for mers-cov on the arabian peninsula) likely contribute to newly diagnosed cases in humans [8] [9] [10] . the potential for continuous reintroduction to humans increases the risk of mers-cov adapting in humans to acquire enhanced human-tohuman transmission profiles, a scenario suspected to have initiated the sars-cov pandemic in 2002-2003 [11] . effective public health measures and culling of civet cats, the zoonotic host for sars-cov, brought the sars-cov pandemic to a rapid end [11] . eliminating mers-cov through culling of infected camel herds is not a practical solution. furthermore, detection of pre-emergent mers-cov-like, and sars-cov-like, strains circulating in bat species indicate that the natural environment is ripe for future human exposures to potentially pathogenic coronaviruses [12] [13] [14] . therefore, the development of therapeutic countermeasures that can interfere with mers-cov pathogenesis is critical to break zoonotic-to-human and human-to-human transmission cycles that may instigate global spread. evaluating the toxicity and efficacy of novel mers-cov therapeutics require the availability of animal models that effectively recapitulate mers-cov pathogenesis during fatal cases of human infections. therefore, the first question in generating a mers-cov animal model would be: what are the pathological features of a human infection? limited histopathological findings from human autopsies indicate that fatal cases of mers-cov results from pneumonia initiated by infection of bronchiolar and alveolar epithelia of the lower respiratory tract (lrt) [15, 16] . pneumonia in the lrt is also the prominent finding on radiographs from x-rays and cts of diagnosed human cases [17] . high viral loads in tracheal aspirates from patients are also associated with severe pulmonary disease [18] , which is indicative of actively replicating mers-cov in the lrt. initial evaluation of the human mers-cov emc/2012 isolate in rhesus macaques demonstrated replication in the lrt with mild pneumonia-like disease ( fig. 1 ) [28] . achieving respiratory pathology reflecting a lethal human disease proved to be more complicated in nonhuman primates. severe respiratory disease in the marmoset produced clinical endpoints consistent with fatal disease that required euthanasia ( fig. 1) [29, 30] . evaluation of two human isolates, jordan and emc/2012, and a tissue cultureadapted mers-cov strain (mers-0) in nonhuman primates resulted in mild disease in rhesus macaques or marmosets ( fig. 1 ) [31] [32] [33] , confounding the reproducibility of near-lethal disease in nhps. nonhuman primates are central to late-stage preclinical evaluation of therapeutic countermeasures, but may be impractical for initial preclinical studies. a small animal model may be applicable if there is limited therapeutic available for toxicity and efficacy testing, especially if large animal numbers are needed to determine confidence and reproducibility. early studies in mouse, hamster, and ferret revealed that conventional small animal models were fully resistant to mers-cov infection and replication (fig. 1) [19, 20, 36] . a seminal study identifying the mers-cov receptor as human dipeptidyl peptidase iv (hdpp4) [49] , and publication of the crystal structure of hdpp4 interacting with the receptor binding domain (rbd) of the mers-cov spike protein [50] , exposed tropism determinants critical for susceptibility. dipeptidyl peptidase iv contact amino acids at the hdpp4/rbd interface are highly conserved among mers-cov-susceptible mammalian species (human, camel, and specific events since the emergence of mers-cov in 2012 are emphasized above the timeline. references to mammalian models evaluated for mers-cov pathogenesis comprise hamster [19] , ferret [20] , rabbit [21] [22] [23] [24] , camel [25] [26] [27] , nonhuman primates [28] [29] [30] [31] [32] [33] [34] [35] , and mouse [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] bat) (fig. 2) [51] . although mouse, hamster, ferret, and guinea pig dpp4 orthologs exhibit high overall similarity to hdpp4, specific amino acid differences at the dpp4/rbd interface account for the inability of these species to support infection [51] [52] [53] [54] [55] [56] . overexpression of a mouse dpp4 (mdpp4) with changes in the contact residues at the dpp4/rbd altered cellular profiles from resistant to susceptible to mers-cov infection [52, 53, 56] . the dependence on dpp4-specific contact was further substantiated by similar studies evaluating modified dpp4 orthologs from the hamster, ferret, and guinea pig [55] . dipeptidyl peptidase iv was identified as the major determinant of mers-cov tropism. researchers rapidly leveraged knowledge of the dpp4 receptor to generate susceptible small animal models ( fig. 1 ) [12] . zhao et al. utilized a unique approach for producing susceptible mice that could replicate human isolates of mers-cov in the lungs by infecting mouse lungs with an adenovirus that constitutively expresses the full-length hdpp4 gene ( fig. 1) [37] . transient expression of hdpp4 supported infection and replication with human strains of mers-cov in the lungs and indicated that this technology may be an effective rapid response platform for initial (c) zoomed-in view of the human dpp4 structure (light gray) with highlighted mers-cov rbd contact residues (dark gray). species-specific contact residues that differ from human are highlighted in red evaluation of emergent and pre-emergent viruses. however, pathology associated with a fatal mers-cov infection was not observed in the ad-hdpp4 model [37] , which limited the capacity to evaluate the efficacy of therapeutic countermeasures. genetic engineering of mice would be necessary to develop preclinical mers-cov mouse models with respiratory phenotypes that reflected clinical outcomes in patients. knock-in of full-length hdpp4 rendered mice susceptible to human isolates of mers-cov at low infection doses ( fig. 1 ) [38] [39] [40] . knock-in mice exhibited severe pulmonary pathology and increased mortality; however, widespread constitutive expression of full-length hdpp4 resulted in high levels of mers-cov infection and replication in extrapulmonary tissues [38] [39] [40] . in some studies, higher viral loads could be detected in the brain compared to the lungs [39, 40] . mice with infections of the central nervous system (cns) exhibited encephalitis that corresponded with the kinetics of mortality [39] . currently, there is no evidence to support a cns component associated with mers-cov pathogenesis in humans. attempts to restrict hdpp4 expression to epithelial cells of the lungs using constitutive tissue specific promoters (e.g., cytokeratin k18) yielded outcomes similar to those observed with sars-cov mouse models, wherein high levels of mers-cov infection/replication were detected in the brains (fig. 1 ) [39] . to circumvent confounding problems associated with global bio-distribution of overexpressed hdpp4 receptor, researchers engineered mouse models using sophisticated molecular approaches. pascal et al. employed regeneron's velocigene technology to replace sequences encoding nearly the entire mdpp4 genomic region with those encoding the exons/introns from the hdpp4 genetic region ( fig. 1 ) [41] . retaining the mdpp4 5 0 and 3 0 genetic elements that regulate expression maintained inherent expression profiles of full-length hdpp4 in mice [41] . importantly, mers-cov infection/replication was readily detected in the lungs with little involvement of extrapulmonary tissues [41] . infection with human isolates of mers-cov caused moderate respiratory pathology with mortality determined by euthanasia of mice at 20% weight loss [41] . unfortunately, commercial restrictions limit the availability and use of this model to the broader scientific community. in addition to the concerns raised above, the first generation of mouse models was developed with the full-length hdpp4, which may alter the inherent physiological properties of the mouse. the multifaceted involvement of dpp4 in maintaining immune homeostasis is of significant importance regarding susceptibility to infectious disease [57] . dpp4 exists in two forms: (1) a membrane anchored form on the surface of multiple cells types (e.g., b cells, t cells, nk cells, and epithelial cells to mention a few) and (2) a secreted form that can be identified in human serum [57] . dpp4 interacts with and modifies heterologous protein molecules involved in nociception, neuroendocrine function, metabolism, cardiovascular function, immune regulation, and infection [57] . modification of heterologous protein function can proceed through cleavage of n-terminal amino acids through the enzymatic activity of the α/b-hydroxylase domain, or allosteric interaction/ signal transduction [57] . the species specificity of dpp4 is exemplified by the interaction of hdpp4 with adenosine deaminase (ada), a well-recognized binding partner of hdpp4, which modulates downstream t cell functions [58] [59] [60] . the hdpp4/ada interaction evolved in higher mammalian species (human, nhp, bovine, rabbit), but not in mouse or rat [58] [59] [60] . interestingly, in one study ada was demonstrated to block infection of mers-cov in tissue culture [20] , indicating that the binding site on hdpp4 for ada, and the mers-cov rbd, may overlap. consequently, introducing full-length hdpp4 into mice may skew innate immune mechanisms that could influence responses to therapeutic countermeasures. in the second generation of mers-cov-susceptible mouse models amino acid residues predicted to function at the mdpp4/ mers-cov rbd interface were modified to avoid the introduction of full-length hdpp4 ( fig. 1) [42, 43] . li et al. recently developed a mouse model wherein the mdpp4 genomic region encompassing exons 10-12 were replaced with the respective genomic region from hdpp4, referred to as an hdpp4 knock-in model (hdpp4-ki) [43]. exons 10-12 encode contact amino acids at the hdpp4/mers-cov rbd interface that were able to support replication of human mers-cov isolates in the lungs, but did not elicit a mortality phenotype [43] . adaptive evolution of human mers-cov in the hdpp4-ki mouse resulted in mouseadapted viruses that evoked a lethal respiratory phenotype with little involvement of extrapulmonary tissues. the lethal respiratory phenotype is a consequence of novel mutations acquired during adaptive evolution. a combination of mutations in both the s1 and s2 regions of the mers-cov spike protein facilitated a lethal respiratory phenotype [43] . results in the hdpp4-ki model substantiate an earlier mouse model referred to as the 288-330 +/+ model, which was designed with only two amino acid changes in mdpp4 to generate mers-cov susceptible mice. genetic engineering and implementation of the 288-330 +/+ mouse model, combined with mers-cov adaptive evolution, is the subject of this chapter. initial studies in tissue culture revealed that human and rodent cell types were resistant to mers-cov infection upon overexpression of mdpp4; however, overexpression of hdpp4 conferred permissivity to infection/replication [53] . comparative structural modeling of hdpp4 and mdpp4 revealed putative contact residues in mdpp4 amenable to modification at the dpp4/rbd interface. modification of two amino acids (a288l and t330r) was sufficient to endow mdpp4 with the capacity to mediate mers-cov infection/replication [53] . shortly after the emergence of mers-cov into humans in 2012, the crispr/cas9 genome editing technology became available for applications to modify mammalian genomes in vitro and in vivo [61] [62] [63] . recognizing our unique situation, we designed crispr/cas9 targets to modify the mouse genome encoding amino acids a288 and t330 in exons 10 and 11 of the mdpp4 gene (fig. 3) [12, 42] . concomitant with mouse development, in vitro studies were initiated to adapt mers-cov to the modified mdpp4 [42] . tissue culture adaption resulted in mers-0 virus, which contained an rmr insertion and s885l mutation in the s2 region of the mers-cov spike protein [42] . a mers-0 molecular clone exhibited enhanced replication kinetics and higher titers compared to human mers-cov isolates. additionally, the mers-0 virus replicated to higher levels in the lungs of 288-330 +/+ mice, compared to human and camel mers-cov isolates [42] . based on these data the mers-0 virus was used to initiate passaging in mice heterozygous for mdpp4 with a288l and t330r mutations, 288-330 +/à (fig. 4) . we reasoned that adaptation around one expressed copy of the mdpp4 with 288-330 mutations, and a wild-type mdpp4 expressed copy, might cultivate generation of a mouse-adapted mers-cov that could utilize wild-type mdpp4 as the primary receptor. after 15 passages we obtained a mouse-adapted mers-cov (mers15c2) exhibiting a lethal respiratory phenotype in the 288-330 +/+ mice [42] . our mers-cov reverse genetic system was used to generate an infectious clone of the mouse-adapted virus, icmersma1 [42] . lethal respiratory pathology with icmersma1 required high infectious doses (5 â 10 6 pfu). an additional 20 passages of icmersma1 in 288-330 +/à mice bore a novel mouse-adapted mers-cov that produced lethal respiratory disease at doses of 5 â 10 5 pfu, and lung pathology associated with severe respiratory disease at 5 â 10 4 to 5 â 10 5 pfu [44] (fig. 1) . this mers-cov model system (288-330 +/+ mice and mouseadapted mers-cov viruses) is now being employed to: (1) understand complex virus-host interactions [12, 31, 42, [64] [65] [66] [67] , (2) evaluate antibody-based therapeutics [42] , (3) evaluate drug-based therapeutic countermeasures [68] , and (4) evaluate anti-mers-cov vaccines [42, 66] . the goal of this chapter is to provide an outline of how to rationally design a mouse with altered susceptibility to mers-cov. for additional information there are a number of detailed reviews and book chapters describing the design and utilization of the crispr/cas9 technology for generating mouse models [69, 70] . . agarose gel separation based on size allows for discrimination between target dna, cas9 digested targets, and guide rnas. (b) schematic utilizing crispr/cas9 technology to genetically engineer mice. fertilized c57bl/6 j zygotes are collected and injected with rna encoding cas9, dpp4 single guide rna, and oligos to facilitate homology-directed repair (hdr). microinjected zygotes are implanted into pseudopregnant recipient female c57bl/6 j mice. offspring are screened by sequencing for the intended change at positions 288 and 330. mice identified as having the appropriate changes are backcrossed to c57bl/6 j mice to maintain the pure c57bl/6 j background, or may be crossed to any desired strain (e.g., balb/cj or 129s1/svimj). (c) table describing sequences of cas9 guide rnas and oligos for hdr to genetically engineer amino acid changes at position 288 (ala to leu) and 330 (thr to arg). (d) sequencing chromatograms highlighting how the f0 offspring from embryo implantation can be a mosaic of insertion/ deletions (indel's) generated by random non-homologous end joining from cas9 cutting at the genomic alleles, and the hdr repair that incorporates the intended changes encoding amino acids at positions 288 and 330. pure homozygous 288-330 +/+ lines were obtained by backcrossing onto c57bl/6 j mice. the highlighted mutations caa (ttg in the reverse orientation) and aga encode the novel 288 l and 330r amino acids 9. after a predetermined number of passages the region encoding the spike protein of mers-cov is sequenced using rt-pcr to amplify the region of interest followed by standard sanger sequencing (see note 6). 10. after 10 passages viruses were plaque purified by diluting the heterogenous stock of virus 10 à1 to 10 à6 , and infecting a monolayer of vero ccl81 cells similar to a standard plaque assay. 11. single plaques are isolated using a pipet tip and the virus expanded on a freshly seeded monolayer of vero ccl81 cells. 12. virus stocks are generated, viral rna is isolated using standard trizol purification, and the region encoding the mers-cov spike protein is amplified by standard rt-pcr techniques and sequenced using standard sanger sequencing. 13 . mutations identified by sequencing must be confirmed using a reverse genetic system to generate an infectious clone encoding the identified mutations [72] . cockrell (fig. 3) the details for generating and using the crispr/cas9 system to generate mutations are outlined in the materials and methods by cockrell et al. [42] . notably, the 288-330 +/+ mice were initially generated in the animal models core facility at the university of north carolina at chapel hill. the extensive technical expertise required for genetic engineering of mice is the subject of many expert reviews and book chapters that will not be covered here. nevertheless, we provide a conceptual overview of the steps to generate the 288-330 +/+ mice. 1. design guide rnas to target each of the a288 and t330 alleles. cockrell et al. designed the guide rnas to direct the cas9 to cut as near the mutation site as possible (fig. 3 ) (see note 7 for helpful resources to design and genetically engineer mouse knockouts). 2. test guide rnas in vitro for the capacity to cut a target sequence (fig. 3) . (a) generate double-stranded odns or a plasmid containing the target sequence with the correct pam site. (b) assemble ribonucleoprotein (rnp) complexes according to manufacturer's instructions (see note 8). (c) subject double-stranded dna with target sequence to rnp complexes and assess digestion pattern on an agarose gel (fig. 3 ). 3. two separate oligos are also designed to introduce the novel mutations on exons 10 (a288l) and 11 (t330r) of mdpp4, through homology-directed repair (fig. 3 ). 4. fertilized zygotes are collected from c57bl/6j female mice that were superovulated and mated to male c57bl/6j mice. cas9 endonuclease, combined with odns encoding the replacement alleles for 288 and 330 in mdpp4, were all pronuclear injected into the fertilized zygote [42] (fig. 3) . the fertilized zygotes were from c57bl/6j mice. 6. the injected embryos were implanted into pseudopregnant recipient females. 7. newly born pups were screened for the presence of the correct change at the 288 and 330 alleles by standard pcr amplification and sanger sequencing (fig. 3 ) (see note 9). 3. 288-330 +/+ mice are brought into the bsl3 laboratory 7 days before start of the experiment to allow for environmental acclimation. 4. mice are anesthetized via intraperitoneal injection of 50-100 μl of a ketamine (50 mg/kg)/xylazine (15 mg/kg) mixture (see note 10). level of anesthesia is assessed by pedal reflex. 6. measure initial weight (day 0 weight) while waiting for mouse to be anesthetized (see note 11). 7. once a pedal reflex is no longer triggered, mice should be immediately infected by the intranasal route. holding the animal vertically, apply 50 μl of virus solution by pipetting onto their nostrils and allow them to inhale. to ensure that all of the 50 μl reach the lower respiratory tract hold the mouse upright for an additional 30 s (see note 12). 8. note any inconsistencies during infection, including: (1) presence of bubbles of inoculum from nasal cavity, (2) occurrence of inoculum in mouth, or (3) failure to inhale entire dose of inoculum. notes will help to explain potential inconsistencies in readout parameters and may be used as exclusion criteria for inefficient infections. 9. mice are put back into the cage and placed on their back to ensure virus solution will stay in the lungs. note: cages are returned to cage rack, but the respiration of mice is continuously monitored by observing breathing. 10. place mice next to each other to keep body temperature as close to normal as possible. 11. check cage after 30-45 min to confirm that all mice wake up from anesthesia and infection. adaptation of mers-0 in 288-330 +/à mice (fig. 4) 1. mouse adaptation was initiated in heterozygous 288-330 +/à mice by infecting with 50 μl of the mers-0 infectious clone (see note 13). 2. at 3 days post-infection the mouse is euthanized by extended exposure to isoflurane.~2 ml of isoflurane is added to the bottom of a jar that can be firmly sealed (see note 14). a thoracotomy is performed to expose the lungs. 4. lungs are removed and placed in a 2 ml gasket sealed skirted screw cap tubes. tubes are previously prepared with 1 ml of 1â pbs containing~5-10 mm of glass beads. lungs are homogenized for 60 s in a bead homogenizer. 6. lung lysates are centrifuged in a microcentrifuge for 5 min at max speed to pellet debris. 7. this is considered passage 1 (p1) and 50 μl of lung homogenate is used to infect a naïve 288-330 +/à mouse. 8. the process is repeated for a desired number of cycles. 1. after infection mice are monitored daily for weight loss for the entire duration of the experiment. 2. to record daily weights, pick up mice by the tail, identify by ear notch, and place into cup on a scale. record weight and calculate percentage of starting body weight (see note 15). 3. mice can also be monitored to determine if they are moribund using a clinical scoring scale whereby: 0 ¼ no clinical signs; 1 ¼ ruffled fur; 2 ¼ ruffled fur with hunched posture (only slight with no signs of dehydration); 3 ¼ as defined in number 2 with more severe signs of dehydration and some loss of body strength; 4 ¼ pronounced dehydration and prominent loss of mobility; 5 ¼ unresponsive to stimuli and prominent eye squinting. 4. it is important to note that weight loss might not always be the most appropriate parameter and animals should be euthanized at the discretion of the researcher even if animals have not reached 80% of their starting weight. mice that approach 80% of their starting body weight (20% weight loss) are euthanized via isoflurane overdose followed by a secondary euthanasia method (thoracotomy or cervical dislocation). depending on the experimental circumstances an institutionally approved exception may be implemented to allow continuation of the experiment (increasing the frequency with which the mice are monitored will likely need to be implemented) (see note 16). 3. respiratory function can be performed every day over the entire course of infection, or on single selected days. investigating a novel respiratory virus may require the investigator to perform a time course to determine the most effective time points for measurement. the largest differences between groups typically correlate with peak viral replication. 4. at each time point measured the mice need to be randomized into different chambers to avoid technical artifacts (e.g., a mouse measured at day 1 in chamber 1 should be evaluated in a different chamber on day 2 measurement). practical considerations dictate that 8-12 animals can be measured at any one time, and each group of 8-12 mice may take an hour for a proficient technician. therefore, experiments should be carefully planned to limit the number of mice to be evaluated. 5 . mice that are difficult to handle can be slightly anesthetized by applying isoflurane to the chamber in order to remove them from the chamber and return to their cage (see note 18). 6. open thorax, paying attention not to cut into lung tissue. 7. assess lung tissue for reddish discoloration and record severity by applying a number 0 (no hemorrhaging) to 4 (severe hemorrhaging in all lobes of the lungs). 8. harvest lung tissue and place in tubes prefilled with sample type specific solution. 9. put scissors and forceps first into cidecon to remove any residual blood and then into 70% ethanol in order to not crosscontaminate samples. 10. whole lung can be used for one assay or different lobes can be used for different assays. steps 1-6 from subheading 3.7. 2. cut into superior vena cava and collect blood with a pipette. 3. blood is typically transferred to a serum/plasma separation tube that allows for separation of serum/plasma from cells. 4. in the event that it is necessary to harvest cells for flow cytometry analysis or vetscan hm5 analysis, the blood sample can be transferred to a tube containing edta. note: vetscan hm5 is a veterinary diagnostic machine that analyzes basic immune cell counts and additional hematological parameters within 2 min. 5. all vetscan assays are performed under bsl3 conditions. removal of samples for downstream analysis outside of bsl3 conditions require specific inactivation procedures that must be pre-approved by the institutional biosafety committee. 3. this can also be achieved using a readily transfectable human embryonic fibroblast cell line such as hek293t cells and selecting for stably transfected cells. 4. cells can be counted by seeding an extra well, but it is safe to assume that the cell number is approximately doubled after 24 h. 5. this can also be achieved using a microscope to assess plaque size if plaques can be readily detected. 6. since the major determinant of mers-cov tropism is the spike protein, it would be anticipated that mutations having the most significant impact on infection might occur within the gene encoding the spike protein. 7. at the time that these mice were being generated in early 2014, crispr/cas9 reagents were not readily available. additionally, there were few bioinformatics tools available to facilitate guide rna design and off-target potential. in the current research environment crispr/cas9 reagents can be sourced from multiple commercial entities and there are a number of bioinformatics tools to assist with design. addgene is a nonprofit plasmid repository where crispr reagents and resources can be readily obtained (https://www.addgene. org/). additional guidance for generating mice using crispr/cas9 technology can be found in more comprehensive protocols [69, 70] . 8. all relevant reagents and protocols can now be obtained from commercial sources as readily synthesized rnas and purified proteins (e.g., integrated dna technologies). 9 . although a number of pups may be identified to have the correct mutation, many will likely be mosaic for random mutations including insertions/deletions due to the higher efficiency of non-homologous end joining (nhej) after cas9 digestion compared to the desired hdr employed to mediate allele modification. 10. the administered dose will depend on the weight of the animal which should be predetermined the day prior to initiating the experiment. 11. it is not necessary to anesthetize mice for measuring daily weights. 12. it is important that the inoculum reaches the lower respiratory tract for a successful mers-cov infection. 13 . mouse adaptation can be initiated with any mers-cov strain that exhibits some pulmonary replication. 14. mice should never come into contact with the isoflurane. to prevent direct contact, a layer of aluminum foil is placed at the bottom of the jar and this is covered with two additional layers of paper towel. 15 . the percent body weight is typically calculated after leaving the bsl3 environment. therefore, the weight sheets should have the anticipated weights of each animal at 20% weight loss. this will provide a real-time indication of when the mice are approaching the criteria established for humane euthanasia. 16. institutional approval is required for animals to be placed under exception. it cannot be emphasized enough that all animal work should be pre-approved by appropriate university iacuc and ibc committees and should be in accordance with the recommendations for the care and use of animals by the office of laboratory animal welfare at nih. 17. assessing respiratory function using plethysmography under bsl3 conditions requires costly equipment and extensive training prior to use. 18. anesthesia should be avoided prior to measuring lung function to prevent interference with lung function measurements. the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from economic impact of the 2015 mers outbreak on the republic of korea's tourism-related industries costly lessons from the 2015 middle east respiratory syndrome coronavirus outbreak in korea middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission middle east respiratory 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infection and disease cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus haagmans bl (2013) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptorbinding domain complexed with human receptor dpp4 coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection permissivity of dipeptidyl peptidase 4 orthologs to middle east respiratory syndrome coronavirus is governed by glycosylation and other complex determinants host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system direct association of adenosine deaminase with a t cell activation antigen revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function crystal structure of cd26/ dipeptidyl-peptidase iv in complex with adenosine deaminase reveals a highly amphiphilic interface multiplex genome engineering using crispr/cas systems rna-guided human genome engineering via cas9 one-step generation of mice carrying reporter and conditional alleles by crispr/cas-mediated genome engineering the human sodium iodide symporter as a reporter gene for studying middle east respiratory syndrome coronavirus pathogenesis giving the genes a shuffle: using natural variation to understand host genetic contributions to viral infections middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis mers-cov accessory orfs play key role for infection and pathogenesis broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses generating genetically modified mice: a decision guide genome editing in mouse embryos with crispr/cas9 reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses new metrics for evaluating viral respiratory pathogenesis key: cord-349010-n4s8dzgp authors: al-tawfiq, jaffar a.; memish, ziad a. title: update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) date: 2016-12-24 journal: expert rev anti infect ther doi: 10.1080/14787210.2017.1271712 sha: doc_id: 349010 cord_uid: n4s8dzgp introduction: the middle east respiratory syndrome coronavirus (mers-cov) is an important emerging respiratory pathogen. mers-cov resulted in multiple hospital outbreaks within and outside the arabian peninsula. the disease has a high case fatality rate, with the need for a therapeutic option. areas covered: in this review, we provide an overview of the progress in the development of therapeutic strategies for mers. we searched pubmed, embase, cochrane, scopus, and google scholar, using the following terms: ‘mers’, ‘mers-cov’, ‘middle east respiratory syndrome’ in combination with ‘treatment’ or ‘therapy’. expert commentary: there are multiple agents tried in vitro and in vivo. none of these agents were used in large clinical studies. available clinical studies are limited to the use of the combination of interferon and other agents. these clinical studies are based solely on case reports and case series. there are no prospective or randomized trials. there is a need to have prospective and randomized clinical trials for the therapy of mers-cov. however, this strategy might be hampered by the sporadic cases outside the large hospital outbreaks. the middle east respiratory syndrome coronavirus (mers-cov) emerged as an important virus in 2012 and since then has caused multiple outbreaks in hospitals especially in the kingdom of saudi arabia and outside the arabian peninsula [1] [2] [3] . since the emergence of mers-cov, a total of 1800 cases including 640 deaths were reported by the world health organization (who) [4] . due to the increased morbidity and mortality of mers-cov infection, the attention was directed toward the development of prevention strategies and the establishment of therapeutic modalities. an earlier review was based on the severe acute respiratory syndrome (sars) experience and had suggested few possible options for the treatment of mers-cov infection [5] . in this review, we provide an overview of the progress in the development of therapeutic strategies for mers. we searched pubmed, embase, cochrane, scopus, and google scholar using the following terms: 'mers,' 'mers-cov,' 'middle east respiratory syndrome' in combination with 'treatment' or 'therapy. ' we also reviewed the references of each article to further include other studies or reports not identified by the search. we classified the studies into the following categories: in vivo and in vitro studies, animal studies, and human case reports or case series. for clinical studies, we graded the level of the evidence based on the 'oxford centre for evidence-based medicine' [6] . in vitro studies showed variable activity of various agents against mers-cov (table 1) . these agents include: interferon, ribavirin, hiv protease inhibitors (nelfinavir, ritonavir, and lopinavir). interferon is antiviral type i ifn system, a major part of the innate immune response [7, 8] . in vitro studies showed that ifn-β has an ic 50 of 1.37 u/ml and that ifn-β has anti-mers-cov activity of 16-, 41-, 83-, and 117-fold higher than ifn-α2b, ifn-γ, ifn-universal type 1 and ifn-α2a, respectively [9] . in vitro studies showed that ifn-β has a lower ic50 for mers-cov compared to ifn-a2b [9] . ribavirin is a nucleoside analog that is activated by host kinases to a nucleotide [7, 10, 11] . it was shown that in vitro doses of ribavirin required to inhibit mers-cov replications are too high to be achieved in vivo [7, 10] . nelfinavir and lopinavir inhibit mers-cov in vitro [7, 12] . the mean 50% effective concentration (ec50) of lopinavir using vero e6 and huh7 cells was 8.0 μm [13] . camostat and the heptad repeat 2 peptide (hr2p) are two mers-cov fusion inhibitors that were tested in vitro [14, 15] . the fusion inhibitor, camostat, inhibited viral entry into human bronchial submucosal gland-derived calu-3 cells but not the immature lung tissue [14] . the second fusion inhibitor, hr2p, inhibits mers-cov replication and the spike protein-mediated cell-cell fusion [15] . cyclosporin affects the function of many cyclophilins that act as chaperones and facilitate protein folding [16, 17] . in vitro, cyclosporine inhibited mers-cov replication [16, 17] . nitazoxanide, a broad-spectrum antiviral agent, and teicoplanin, an inhibitor of cathepsin l in the late endosome/lysosome and blocker of the entry of mers-cov, also showed inhibitory effect of mers-cov in vitro [18, 19] . there are few studies evaluating various agents as therapy for mers-cov in animal models (table 2) [12, [21] [22] [23] [24] . in the rhesus macaques model, interferon-α2b-ribavirin combination decreased viral replication within 8 h of mers-cov infection [25] . in a primate model, the mortality rate at 36 h post-inoculation was reduced from 67% in untreated to 0-33% in animals treated with a combination of interferon-β1b and either lopinavir or ritonavir [12] . intranasal use of an hr2p analog with improved pharmaceutical property, hr2p-m2, was protective in mice model [21] . in an animal model using mers-cov infected mice, the use of high titer mers immune camel serum was effective in reducing lung injury and acceleration of virus clearance [22] . mycophenolate has a direct and indirect antiviral activity by modulation of ifn response [20] . the use of mycophenolate in the common marmoset animal model resulted in higher mortality than untreated animals [12] . a monoclonal antibody designated as m336 is an antibody derived from a large phage-displayed antibody library from b cells of healthy donors [26] . the use of this m336 in mice showed promising results as a therapeutic and a prophylactic agent [23] . currently, there is no animal model that completely reflects the course of mers-cov disease in humans and thus the data obtained from these animal models are to be interpreted cautiously. and animal models utilize therapy shortly after infection. based on analysis of sars data, interferon-ribavirin combination was suggested as a possible therapeutic option for the treatment of mers-cov infections [5] . limited data are available regarding the clinical efficacy of antiviral agents [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] ( table 3 ). the first use of the combination of ribavirin-interferon therapy was in five patients with mers infection [27] . the therapy was started late in the course of the disease with a median time from admission to therapy of 19 days [27] . of the included 5 patients, none responded to therapy [27] . in a subsequent retrospective cohort study, 20 mers patients received ribavirin-interferon compared to 24 patients who did not [28] . the 14-day survival rate was better in those who received the combination therapy (70% vs. 29%, p = 0.004); however, the 28-day survival rate was not statistically different (30% vs. 17%) ( table 1 ) [28] . in another case series, 11 mers patients had ribavirin and peg-interferon α-2a [29] . ribavirin-ifn-α2a was compared to ribavirin-ifn-β1a in a study of 13 and 11 patients, respectively [30] . the mortality rate was not statistically different between the two groups (85% vs. 64%) [30] . in a large cohort study of 51 patients, various combinations of interferon and ribavirin were used with different outcomes (table 3 ) [31] . in a case series of 6 patients, 3 patients received ribavirin and interferon-alfa 2b within 1-2 days of admission and they survived compared to the other 3 patients who died as they received the therapy 12-19 days after admission [36] . another study evaluated the use of interferon beta, interferon alpha, or ribavirin and showed survival rates of 18/23 (78.3%), 6/8 (75%), and 13/19 (68.4%), respectively (table 3 ) [31] . the combination therapy was also used in other case reports (table 3 ) [33, 34] . the role of the combination of ribavirin and ifn was also tried as a treatment and a prophylaxis [34] . the current studies of the use of ribavirin and ifn combination therapy for mers-cov infection rely on small number of patients but there is a trend for improvement. thus, it was suggested that the combination of type 1 interferon and ribavirin could be used [37] . due to the inhomogeneous nature of available studies and the limited data that are available, a precise recommendation on therapy of mers could not be established. the combination of lopinavir/ritonavir, ribavirin and interferonalpha was used in one case [32] . one patient received pegylated interferon, ribavirin, and lopinavir/ritonavir from day 13 of illness and the patient had continued mers-cov in the respiratory tract secretions until the fourth week of illness [33] . however, viremia was detected for only 2 days after initiation of triple therapy [33] . in a case series, eight patients received mycophenolate mofetil and all survived [31] . in the sars epidemic, passive immunotherapy with neutralizing antibodies was considered as a therapeutic approach. there are multiple antibodies against mers-cov [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] (table 4 ). in the mers-cov infection, the production of large quantities of mers-cov neutralizing human polyclonal antibodies was possible using gamma-irradiated whole-killed virion vaccine or a spike protein nanoparticle vaccine in a bovine model [49] . utilizing one dose of these antibodies prevented infection in mice [49] . these antibodies were effective when given 12 h before or 24 and 48 h after mers-cov infection [49] . corti et al. isolated a potent mers-cov-neutralizing antibody (lca60) from memory b cells of an infected individual. the lca60 antibodies bind to a site on the spike protein and neutralize mers-cov infection [48] . these lca60 antibodies were used successfully in mice model [48] . similarly, utilizing a humanized mouse model of mers-cov infection, antibodies against the spike protein were efficacious as prophylaxis [39] . antibodies obtained from the sera of mers immune camels were supportive of the clearance of the virus, and reduction of the severity of the disease in mers-covinfected mice [22] . however, the purification and safety of these antibodies in humans has not been established yet. the cellular dipeptidyl peptidase iv (ddp iv; known as cd26 or adenosine deaminase (ada)-complexing protein-2) [48] is an important receptor that mediates mers-cov infection through the viral spike (s) protein [46, 50] . the mers-cov receptor binding domain (rbd), present on the surface spike protein (s), binds to the host cells receptor dpp iv [46, 50, 51] . in humans, dpp iv is present mainly on the lower respiratory tract area such as the bronchial epithelial and alveolar cells [52, 53] . although ddp iv is important for the viral entry into host cells, the use of dpp iv inhibitors, sitagliptin, vildagliptin, and saxagliptin, does not block the infection of mers-cov [50] . in vitro use of monoclonal antibodies (mers-4) exhibited ic50 of 0.056 µg/ml [43] . other possible human mab (m336, m337, and m338) neutralize pseudovirus and live virus [45] . in rhesus model of mers-cov infection, a human monoclonal antibody, 3b11-n, against mers-cov was effective in reducing the pathology of mers-cov [47] . the use of polyclonal antibody (pab) against cd26 inhibits mers-cov infection in vitro [50] . humanized anti-cd26 monoclonal antibodies (mab) such as mab ys110 and 2f9 significantly inhibit mers-cov infection in vitro [38] . polyclonal antibodies against the mers-cov s1 domain neutralize the virus infection [46] . many other mers-cov antibodies are being developed and tested [38] . in the sars epidemic, convalescent plasma was thought to improve the outcome of sars patients [5] . previous studies suggest that convalescent plasma may be used for patients with sars and severe influenza and may result in decreased viral load and a lower mortality rate [54] [55] [56] [57] . however, most of the studies were of low or very low quality, lacked control groups, and had risk of bias [58] . two patients with mers-cov infection received intravenous immunoglobulin in an attempt to treat the infection, one patient was in saudi arabia [59] and the other was in the usa [60] . a protocol for the use of convalescent plasma as a therapeutic option for mers was suggested [61] . plasma donors were identified as those with anti-mers-cov indirect immunofluorescence assay (ifa) antibodies (titer of ≥1:160) with no evidence of active mers-cov infection [61] . in nine confirmed survivors of mers-cov infection, 55%, 33%, and 22% of them had positive mers antibodies by ifa at 3, 10, and 18 months, respectively [62] . the two patients who had long lasting antibodies had severe disease; however, the titer of the ifa antibodies was not measured in the study [62] . in a larger study, mers-cov neutralizing antibodies were produced at low levels and were short-lived [63] . further studies of the kinetics of the mers-cov antibodies showed that all surviving patients and 50% of fatal cases produced igg and neutralizing antibodies [64] . the presence of antibodies did not lead to the elimination of virus from the lower respiratory tract [64] . in a study of 12 patients from south korea, nine patients had prnt50 titers >1:320 by day 21 and two had titers >1:320 by day 28 [65] . in a study of 443 samples, 12 (2.7%) had reactive elisa results, and 9 of those had reactive indirect fluorescent antibody and microneutralization assay titers [66] . thus, the use of convalescent plasma for the treatment of mers-cov in a clinical trial may be challenging due to a small pool of potential donors with sufficient antibody titers [66] . based on sars experience, some authors suggested that steroid therapy might be beneficial for severe mers-cov infection [67] . corticosteroid use for patients with sars showed that early use of corticosteroids significantly increased viral load, and 20.7-fold increase in risk of icu admission and mortality [68] . in another study of 16 non-icu patients, the median time for sars-cov to become undetectable in plasma was 12 days compared to 8 days in patients who did and did not receive early corticosteroid therapy [69] . corticosteroids were used as adjunct therapy for many patients with mers-cov [27, 70] . in a study of 13 patients with mers-cov infection, one patient received steroid and intravenous immunoglobulin for thrombocytopenia [59] . initial study of five patients, three patients received a combination of interferon and ribavirin in addition to adjunct steroid on day 0-21 and all died during hospitalization [27] . however, steroids were not evaluated systematically as therapy for mers patients. the host protease, furin, is an important factor to break down the s1-s2 region of the mers-cov [71] . in vivo studies showed activity of multiple agents against mers-cov and include: chloroquine, chlorpromazine, cyclosporine, and mycophenolic acid [7, 72] . in addition, the us fda approved repurposed agents with broad antiviral activity including in vitro activity against mers-cov [73] . these agents include the polymerase inhibitor bcx4430 and the helicase inhibitor ssya10-001, spike binding (immunoadhesin (dpp4-fc)) [73] . there are many other agents currently in preclinical investigation such as: fluspirilene, thiothixene, fluphenazine hydrochloride, promethazine hydrochloride, astemizole and chlorphenoxamine hydrochloride [72] . the emergence and continued cases of mers-cov infection require the availability of mers therapy. there is an urgent need for the development of standardized animal models and the establishment of standardized clinical therapeutic protocols. the current clinical studies are limited to case reports and case series with no control arm. the quality of evidence these studies offer is too low to make a conclusion. it is difficult to draw conclusion in the face of these limited studies. the most used combination of therapy was interferon and ribavirin as developed initially in 2013. the development of novel therapeutic agents or the repurposing old therapeutic agents against mers-cov are needed as alternative pathways for testing and clinical trials. it was thought that convalescent sera may provide an exciting alternative as a therapeutic agent; the present data does not support the wide adaptation of this therapy. the current mers therapy relies on supportive care and providing circulatory and ventilation support. it is expected that the development and the use of repurposed drugs would allow the development of therapeutic agents for mers-cov. the best location to provide a randomized controlled trail was thought to be the intensive care units for the use of convalescent sera; the presence of milder disease may necessitate the development of therapeutic protocols for patients with severe and those with milder disease. monoclonal and polyclonal antibodies may offer further therapeutic options for the disease in humans. since the prospect for a randomized clinical trial is low due to the sporadic nature of the disease outside hospital outbreaks, it is prudent to have well-conducted prospective clinical studies. the proteins involved in mers-cov entry and replication are attractive targets for the development of antiviral therapeutics. clinical studies utilizing anti-mers-cov antibodies as therapeutic options would add to the prospect to develop therapeutic agents for this syndrome. although many drugs appear to be effective in vitro, a consideration of their availability, pharmacokinetic/pharmacodynamic properties, and side effects should be taken into consideration. of medications with an attractive use, lopinavir, interferon, and mycophenolate are among these agents. accelerated and preferably randomized controlled trails should be conducted. neutralizing antibodies are also promising and the use of these agents in humans. targeting the ddp4 receptor may be of particular importance; however, it is important to keep in mind that the development of any mutation in the binding sites may limit the use of these agents [74] . few monoclonal antibodies showed protective efficacy as a prophylaxis in animal models [39, 48] . the development and testing of monoclonal antibodies are associated with high costs and lack of an undefined 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survivors transmission of merscoronavirus in household contacts viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection •• a study of viral shedding of mers-cov kinetics of serologic responses to mers coronavirus infection in humans feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia clinical recommendations from an observational study on mers: glucocorticoids was benefit in treating sars patients the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mechanisms of coronavirus cell entry mediated by the viral spike protein repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection development of medical countermeasures to middle east respiratory syndrome coronavirus towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for middle east respiratory syndrome coronavirus (mers-cov) • a study of use of human neutralizing monoclonal antibodies for mers-cov research and development activities for middle east respiratory syndrome: the current landscape. n d the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. key: cord-323428-jd91k19z authors: ababneh, mustafa; alrwashdeh, mu’men; khalifeh, mohammad title: recombinant adenoviral vaccine encoding the spike 1 subunit of the middle east respiratory syndrome coronavirus elicits strong humoral and cellular immune responses in mice date: 2019-10-11 journal: vet world doi: 10.14202/vetworld.2019.1554-1562 sha: doc_id: 323428 cord_uid: jd91k19z background and aim: middle east respiratory syndrome coronavirus (mers-cov) has rapidly spread throughout the middle east since its discovery in 2012. the virus poses a significant global public health threat with potentially devastating effects. in this study, a recombinant adenoviral-based vaccine encoding the spike 1 (s1) subunit of the mers-cov genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. materials and methods: mice were immunized initially by intramuscular injection and boosted 3 weeks later by intranasal application. expression of the s1 protein in the lungs and kidneys was detected using conventional polymerase chain reaction (pcr) and immunohistochemistry (ihc) targeting specific regions within the s1 subunit at weeks 3, 4, 5, and 6 after the first vaccination. antigen-specific humoral and cellular immune responses were evaluated in serum and in cell culture following in vitro stimulation with a specific 9-mer epitope within the s1 protein (cysslildy). results: s1 protein expression was only detected by ihc in the kidneys of the ad-mers-s1 group at week 6 from first immunization, and in both lungs and kidneys of ad-mers-s1 group by conventional pcr at weeks 3 and 5 post-prime. the vaccine elicited a specific s1-immunoglobulin g antibody response, which was detected in the sera of the vaccinated mice at weeks 4 and 6 from the onset of the first immunization. there was a significant increase in the amount of th1-related cytokines (interferon-γ and interleukin [il] 12), and a significant decrease in the th2-related cytokine il-4 in splenocyte cell culture of the vaccinated group compared with the control groups. conclusion: the results of this study suggest that this recombinant adenovirus vaccine encoding the s1 subunit of mers-cov elicits potentially protective antigen-specific humoral and cellular immune responses in mice. this study demonstrates a promising vaccine for the control and/or prevention of mers-cov infection in humans. middle east respiratory syndrome coronavirus (mers-cov) is a newly emerging human coronavirus that was discovered in 2012 in a 60-year-old saudi arabian man [1] . following its discovery, many cases were identified in different regions of the arabian peninsula and worldwide thereafter [2, 3] . the most recent outbreak occurred in june 2015 in south korea and was linked to a south korean man who had recently traveled to the middle east [3] . the infection then rapidly spread to 26 persons through close contact in a hospital. within a few months, many cases (n=186) were reported in hospitalized and non-hospitalized persons in south korea [3] . the disease showed a high mortality rate that reached up to 40%, which was higher than that of the severe acute respiratory syndrome coronavirus (sars-cov) outbreak in 2002-2003 (10%) [4] . coronaviruses belong to the subfamily coronavirinae within the family coronaviridae of the order nidovirales [5] . five human coronaviruses were identified (229e, oc43, nl63, hku1, and sars-cov) before mers-cov. lineage c of betacoronaviruses includes bat coronaviruses, which give a primitive impression regarding the origin of the virus [6] . the detection of mers-cov and its neutralizing antibodies in the sera of dromedary camels has shed light on the role of the camel as a possible animal reservoir, which may transmit the virus to humans [7] [8] [9] [10] . indeed, researchers isolated the same mers-cov strain from both a camel "in a barn" and its infected owner in saudi arabia, thus providing further evidence of the potential airborne and direct contact transmission of the virus between camels and humans [11] . there have been several attempts to develop a protective vaccine against mers-cov [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . researchers around the world are focused on the spike protein as the main target for vaccine development against mers-cov. the spike protein of mers-cov attaches to the host dipeptidyl peptidase-4 (dpp4) receptor, which is expressed on several types of human cells [24] . many studies published since 2012 suggesting vaccine models were constructed based on the spike protein and its receptor-binding domain (rbd) region to elicit a strong and protective immune response in vitro and in vivo [25] . recombinant adenoviral vector vaccines are one of the most effective vaccines and showed interesting results during sars-cov outbreaks [12, 26, 27] . since 2013, several studies were published, in which different viral vectors (e.g., adenoviruses and vaccinia virus) were used to develop recombinant vaccine candidates based on full spike gene or part of it and tested their ability to produce protective immunity against mers-cov infection [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . however, further investigations are needed on these suggested vaccines including testing their ability to elicit neutralizing antibodies in different animal models, stimulation of both innate and adaptive immune responses and their corresponding cytokine and chemokine profiles, distribution within the host, and their safety and duration profiles. in this study, a recombinant adenoviral-based vaccine encoding the spike 1 (s1) subunit of the mers-cov genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. all procedures performed in this study were approved by the jordan university of science and technology animal use and care committee. the recombinant human adenovirus type 5 (de1/e3) vaccine was designed to express the s1 domain of the spike protein of mers-cov isolate hu/ jordan-20140010168/2014 (genbank accession number kt861627) under the control of the cytomegalovirus (cmv) promoter. this vaccine was constructed in the laboratories of vector biolabs (the gene delivery company, pa, usa) [28] . six to eight week-old male balb/c mice (n=168) were purchased from animal house at jordan university of science and technology (irbid-jordan) and distributed into three groups in a replicate manner (56 each). animals in the first group were immunized with the ad-cmv-mers-cov-spike1 "ad-mers-s1." animals in the second group received the vector-only "ad-cmv," and animals in the last group served as a control and received 1× phosphate-buffered saline (pbs). for the first immunization, the ad-mers-s1 vaccine was injected intramuscularly at a dose of 0.1 ml containing 1×10 7 pfu/ dose/mouse. the same volume of ad-cmv or pbs was used for the other two groups. the booster dose was carried out 3 weeks later by intranasal route at a dose of 0.1 ml containing 1×10 7 pfu/dose/mouse. all experimental procedures used in this study were approved by the animal care and use committee at the faculty of veterinary medicine (irbid-jordan). lung, kidney, and spleen tissue samples were harvested from each mouse in all groups after 3, 4, 5, and 6 weeks from the first immunization. parts of lung and kidneys were preserved in 10% formalin solution for 24-h, then embedded in paraffin blocks for immunohistochemical analysis, while the other parts were stored at −80°c for polymerase chain reaction (pcr) analysis. spleen tissues were harvested and stored at −80°c for cell culture analysis. blood samples were drawn through facial vein route, left 30 min at room temperature, and centrifuged at 2000 rpm for 20 min. collected sera were stored at −20°c for subsequent analysis. to study the distribution of the recombinant vaccine, total of 50-100 mg of lung and kidney tissues at weeks 3 and 5 post-prime immunization was cut and homogenized in 1 ml of diethyl pyrocarbonate-treated water. homogenized samples were then centrifuged at 800 rpm for 5 min, and 150 µl of the supernatant was used for the extraction. the extraction was performed using the viral gene-spin rna extraction kit (intron biotechnology, korea) according to the manufacturer's instructions. the extracted rna was used for cdna synthesis through a reverse transcription system (promega, madison, wi, usa) according to the manufacturer's instructions. the cdna was kept at −20°c until pcr analysis. the dna template of the constructed vaccine was also purified and used as a positive control in this experiment. heminested pcr was carried out using specific primers targeting specific sequences within the s1 region, as shown in table1 [29] . the first pcr targeted a 1172 bp sequence. pcr mixture contained 0.4 µm of each primer and using 3 µl of the cdna template. touchdown pcr assay was programmed on the mycycler thermal cycler (bio-rad laboratories, hercules, ca, usa) as the following conditions: 95°c for 10 min, followed by 10 cycles of 95°c for 35 s, 63-54°c for 40 s, and 72°c for 1 min and 15 s, then 35 cycles of 95°c for 40 s, 56°c for 40 s, 72°c for 1 min and 15 s, and a final extension step at 72°c for 5 min. two microliters from the first pcr product were used as a template for the second (heminested) pcr to amplify the 1142 bp product following the first pcr conditions. the bands were visualized in 1.5% agarose gels using an ultra-violet transilluminator. localization of s1 protein expression was investigated in both kidney and lung tissues from all groups at week 6 after the first vaccination. ihc using rabbit-specific hrp/dab (abc) detection ihc kit (abcam, cambridge, uk) was adapted, with using antinovel coronavirus spike protein s1 polyclonal antibodies (1:1000; sino-biological, pa, usa). a 5-µm thick section of formalin was fixed, and paraffin-embedded tissues were cut and loaded on specially coated slides. the prepared slides were put in an oven at 70°c for 30 min followed by 15 min in a xylene solution for deparaffinization. the sections were dried and surrounded by liquid blocker (dako pen). the antigen retrieval (pretreatment) step was executed by immersing the sections in 1× reveal decloaker reagent (biocare medical, pacheco, ca, usa) and heating in a microwave several times for 30 min then cool at room temperature for 15-20 min. after that, staining procedure was carried out by following the manufacturer's instructions of the abc kit. to eliminate the effect of internal renal biotin, 0.05% avidin was applied (sigma-aldrich, st. louis, mo, usa) only on kidney sections for 15 min at room temperature [30, 31] . the immunoreactivity of the expressed s1 protein was visualized using a light microscope at different magnifications. spleen tissues from each group were collected, pooled, and homogenized to obtain single-cell suspensions. the cells were centrifuged for 10 min at 800 rpm, and the pellet was re-suspended in roswell park memorial institute medium (rpmi) (gibco, grand island, ny, usa), supplemented with 10% fetal calf serum, 20 mm hepes, 10 µg/ml penicillin/streptomycin, 2 mm l-glutamine, 50 µm 2-mercaptoethanol, and 500 ng/ml amphiostat b (complete rpmi) [32] . the cell suspension was washed again by centrifugation as described earlier. spleen cells were re-suspended in red blood cell lysis buffer (trisbuffered nh 4 cl) to remove erythrocytes. cells from each group (1×10 5 ) were re-suspended in 1 ml of complete rpmi and plated in 24-well plates in triplicate in parallel, adjacent wells. the first set of samples was stimulated with 10 µg of specific peptide "epitope" cysslildy per well. this antigen was defined as the highest potential t-cell epitope from a total of eight potential epitopes within the rbd region of the s1 subunit of the spike protein of mers-cov genome and was expected to display interactions with the maximum number of major histocompatibility complex (mhc) class i molecules, and to show the highest peak in the b-cell antigenicity plot [33] . the second set of samples was stimulated with 50 µg of non-specific stimulant phytohemagglutinin to confirm the viability and productivity of the cultured cells. the third set of samples was not stimulated as a control set. cells were incubated in round-bottom 24-well microtiter plates at 37°c in 5% co 2 . the cultured media were collected after 96 h, and the samples were centrifuged for 20 min at 2000 rpm. the supernatants were stored at −20°c before cytokine analysis, while the pellets were stored at −80°c until real-time (rt)-pcr analysis. detection and measurement of specific mers-s1 igg were achieved using a semi-quantitative anti-mers-cov elisa (igg) (euroimmun ag, luebeck, germany) with a slight modification using universal enzyme conjugate, anti-mouse iggconjugated hrp, to be able to detect mouse igg antibodies instead of human igg antibodies. microtiter plates were pre-coated with purified s1 antigen of mers-cov. mouse diluted serum samples (1:100) were incubated in each well from the three experimental groups (ad-mers-s1, ad-cmv, and pbs) for 30 min at room temperature by following the manufacturer's instructions. the color intensity was measured using an elisa plate reader (biotek, winooski, vt, usa). cell culture supernatants from stimulated and corresponding non-stimulated samples of the three groups were tested for the level of mouse cytokines at weeks 4 and 6 post-prime using a quantitative sandwich enzyme immunoassay technique (quantikine elisa mouse cytokines immunoassay, minneapolis, mn, usa) according to the manufacturer's instructions. relative quantification of mouse ifn-γ and il-4 was normalized to the housekeeping gene β-actin in the cell culture yield of stimulated groups. the collected pellets from cell culture were used to extract total rna using the sv total rna isolation system (promega) according to the manufacturer's instructions; this extraction kit has a dnase incubation step to eliminate any dna in the sample. isolated rna (40 µl) was stored at −80°c before rt-pcr analysis. rna concentration was adjusted to 10 ng/µl using te buffer. purified rna (10 µl) was used as a template to produce 20 µl of cdna using the reverse transcription system (promega) kit. relative rt-pcr assay was performed to determine the mrna expression levels of mouse ifn-γ and il-4 in the cell culture. the fold changes were calculated using the 2 −δδct method normalized to β-actin [34] . specific primer sets were used, as shown in table2 [35] . extracted rna (2 µl) was used as a template in the pcr reaction mixture (20 µl), which was composed of 0.5 µm each of the forward and reverse primers, 10 µl of powerup sypr green master mix (applied biosystems, foster city, ca, usa), and 6 µl of nuclease-free water. rt-pcr conditions were programmed in rotor gene-q (qiagen, hilden, germany) as follows: 50°c for 2 min, 95°c for 10 min, followed by 40 cycles of 95°c s, 60°c for 1 min, and a melting step at 55-99°c for 30 s. data for elisa and fold changes were expressed at mean ± standard deviation. one-way anova and t-test were used to compare different values in all groups using openepi software (emory university, usa). parameter differences were considered statistically significant at p<0.05. parameter differences were indicated by small letters labeled within each group; different letters indicated significant differences at p<0.05. distribution and expression of the s1 subunit of the mers-cov spike protein in mice tissues were detected at weeks 3 and 5 post first immunization in the kidneys and lungs of the vaccinated group but not in control groups using conventional pcr (figure-1 ). using ihc, s1 protein expression was detected in the cytoplasm of proximal tubule epithelium in the vaccinated mice at week 6 post first immunization but not in the control groups (figure-2) . s1 expression was not detected in lung tissues in either the vaccinated or the control groups (figure-2) . there was a significant level (p<0.05) of mers-s1 specific igg antibodies detected in the mice sera of the vaccinated group at weeks 4 and 6 post-prime. the combined results of weeks 4 and 6 revealed that the igg antibody response was significantly higher (p<0.05) in the vaccinated group than in the control groups (figure-3) . available at www.veterinaryworld.org/vol.12/october-2019/7.pdf the level of mouse cytokines at weeks 4 and 6 after the first vaccination in the cell culture supernatants of stimulated and corresponding non-stimulated samples of the three groups was tested by sandwich elisa assay. mice cytokines (ifn-γ, il-12, and il-4) production in the ad-cmv and pbs groups were combined and expressed collectively as one group (control groups) due to the non-significant differences shown across these time points between these two groups (figure-4) . therefore, the levels of each cytokine at the two tested time points (weeks 4 and 6 after the first vaccination) were also combined and expressed as one unit. regardless of in vitro stimulation, production of ifn-γ was significantly (p<0.05) higher in the vaccinated group compared with the control groups (ad-cmv and pbs) at all tested time points (figure-4) . in terms of il-12 production, in vitro stimulation with the mers-specific peptide "cysslildy" resulted in a significant (p<0.05) upregulation in production when compared with the antigen-stimulated and non-stimulated cell culture in the vaccinated group only (figure-4) . although there was no significant difference in il-4 production between the control groups and the vaccinated group in non-stimulated cell culture, antigen-specific stimulation clearly revealed a higher production of this cytokine in the control groups above the vaccinated group level of production (p<0.05). in addition, in vitro stimulation with the mers specific peptide "cysslildy" resulted in a significant upregulation in this production of this cytokine in the control groups while the same antigen stimulation resulted in a significant decrease in il-4 production in the vaccinated group (p<0.05) (figure-4) . at week 4 post first immunization, the fold change in ifn-γ gene expression was significantly higher than that in the control groups (ad-cmv and pbs) (p<0.05). however, there was a prominent increase in ifn-γ gene expression at week 6 postprime in both the vaccinated and ad-cmv groups compared to that in week 4 (p<0.05). the expression of ifn-γ was slightly higher but not statistically significant in the vaccinated group compared to that in the ad-cmv group at week 6 (p>0.05) (figure-5) . the only significant change (p<0.05) in il-4 gene expression was observed in the ad-cmv group at week 6 (figure-5) while no significant changes were observed in the vaccinated group at any time point. in this study, potentially protective humoral and cellular immune responses were elicited in mice by immunization using a recombinant adenoviral-based vaccine encoding the s1 subunit of the mers-cov spike gene. several studies have been published that constructed similar vaccines against mers-cov and tested their ability to induce production of neutralizing antibodies and other immune system components [13, 14, 17, [19] [20] [21] [22] [23] . the results of these studies were encouraging and showed that 12] , and il-4) in vitro. regardless of in vitro stimulation, production of ifn-γ was significantly (p<0.05) higher in the vaccinated group with ad-middle east respiratory syndrome (mers)-s1 compared with the control groups (ad-cytomegalovirus and phosphate-buffered saline) at all tested time points. in vitro, antigen-specific stimulation resulted in a significant upregulation of il-12 production when compared with the antigen-stimulated and non-stimulated cell culture in the vaccinated group only. in vitro stimulation using the mers-specific peptide "cysslildy" resulted in a significant upregulation of il-4 production in the control groups while the same antigen stimulation resulted in a significant decrease in il-4 production in the vaccinated group. ns: non-antigen stimulated, ag: antigen-stimulated. different letters mean significantly different at p<0.05. available at www.veterinaryworld.org/vol.12/october-2019/7.pdf such vaccines may be promising to prevent the global threat of mers-cov infection [13, 14, 17, [19] [20] [21] [22] [23] . however, the safety and efficacy of these vaccines still to be confirmed. it was confirmed that the s protein is responsible for the production of neutralizing antibodies [22, 31] . the s1 subunit contains the rbd, which binds to its host receptor dpp4 (also known as cd26) and induces production of specific neutralizing antibodies. several studies have supported the role of this domain to elicit protective immunity against mers-cov challenge [25, 36, 37] . in this study, we were able to demonstrate the distribution and expression of the s1 protein in the vaccinated group but not in the control groups (ad-cmv and pbs) by detection of the truncated protein-encoding segment in kidney and lung tissues following vaccination using conventional pcr and ihc. these findings support the ability of this vaccine candidate to distribute effectively within host tissues and express the target protein. it has been established that dpp4 is expressed on multiple cell types in the human body including cells in the kidneys and lungs [24] . however, the n glycoprotein, but not the s glycoprotein, of mers was the target of investigation of mers-cov localization using ihc [38] . the n glycoprotein was clearly present in lung tissues following mers exposure. in fact, the current study can be considered pioneering given the use of the s1 glycoprotein to express viral distribution in tissues. therefore, the expression of s1 protein in kidney tissues and not in lung might be used to explain previous in vitro experiments, which found a cytopathic effect of the mers-cov infection inside primary human kidney cells but not in primary human bronchial epithelial cells [39] . in a recent study, the adenoviral vector with enhanced green fluorescent protein but not the adenoviral vector with mers in a hdpp4 mice experiment after intramuscular or intranasal injection [21] . this supporting our finding that this construct was able to reach the lung tissues, but the s1 protein was not expressed in lung tissues. however, s1 gene detection in lung tissues by conventional pcr and not by ihc indicates further investigation is necessary regarding the level and intensity of s1 expression in this tissue. additional studies are also needed to explore s1 protein expression in other tissues such as the liver and intestine given the expression of dpp4 in these tissues [40] . in this study, there was a significant serum level of the s1-specific igg antibody in the vaccinated group but not in the control groups (ad-cmv and pbs). similar results have been shown previously: the sars-cov vaccine had a strong ability to elicit the s1-specific igg antibody response [26] . the results presented in this study also supported the findings of recent publications applying a similar vector vaccine construction [13, 14, 17, [19] [20] [21] [22] [23] . the t helper 1 immune response is known as the dominant immune response in the case of intracellular pathogens, such as viruses and bacteria, while the t helper 2 immune response is the dominant immune response in cases of extracellular pathogens and allergic reactions [41] . many pathogens, especially viruses, shift the host immune response toward th2 dominance over the th1 response to evade cellular immunity. in this study, ad-mers-s1 was able to provoke pro-inflammatory but not anti-inflammatory cytokine release. both th1 and th2 responses were elicited following immunization. cytokines such as ifn-γ and il-12, which represent the th-1 response, were upregulated, while il-4, which represents the th-2 response, was downregulated. the ad-mers-s1 was able to induce production of a significant amount of ifn-γ regardless of antigen-specific stimulation. a similar increase in il-12 was apparent in vaccinated animals in response to specific in vitro antigen stimulation. the mrna expression of ifn-γ was detected as early as 4 weeks after the first immunization. in contrast, il-4 production in cell culture showed a significant increase in control animals after antigen stimulation, while the production of this cytokine was significantly decreased in the at week 4, the fold change in ifn-γ gene expression was significantly higher in the ad-middle east respiratory syndrome (mers)-s1 group than that of both control groups (ad-cytomegalovirus [cmv] and phosphate-buffered saline). however, there was a prominent increase in ifn-γ gene expression at week 6 in both ad-mers-s1 and ad-cmv groups compared to that in week 4. the only significant change in il-4 gene expression was in the ad-cmv group at week 6, while no significant changes were observed in the ad-mers-s1 group at any time point. different letters indicate statistically significant differences at p<0.05. vaccinated group. the results demonstrated neither positive nor negative impact on il-4 expression in mice vaccinated with the ad-mers-s1 or mice that received only pbs. interestingly, there was a significant increase in il-4 and ifn-γ gene expression in antigen-stimulated cell culture obtained from mice vaccinated with ad-cmv at week 6. this might be due to the ability of adenoviruses to elicit strong humoral and cellular immune responses [42, 43] . therefore, it can be concluded that the presence of the s1 protein gene in the adenovirus vector genome disturbs the immune stimulation ability of the adenovirus vector to elicit il-4 cytokine. two studies have shed light on the ability of the s1 subunit, and specifically, the ability of the rbd domain, to shift the host immune response toward th1 [14, 44] . it was demonstrated in these two studies that igg1, which is produced mainly during the th2 immune response, was decreased, while igg2a, which is produced mainly during the th1 immune response, was upregulated following vaccination with their vaccine candidate. the truncated rbd fused with the fc portion of human igg was able to elicit both isotypes with a relatively higher amount of the igg2a isotype (th1 response) [44] . recombinant adenovirus vector carrying the s1 subunit or the whole s gene was also able to induce both types of the immune response. the igg2a level was detected earlier and higher in mers-s than mers-s1 at week 2 post-vaccination but not after week 3 [14] . accordingly, these findings lead to the dominance toward the th1 cellular immunity. several in vivo and in vitro studies have revealed the ability of mers-cov to evade the host innate immune response through downregulating the antigen-presenting pathway and proinflammatory cytokines such as ifn-γ and il-12 [38, [45] [46] [47] . several mers-cov proteins were previously identified as potent ifn antagonists, including the m structural protein and the orf 4a, orf 4b, and orf 5 accessory proteins [46] . in addition, a comparison of the immune response of two mers-infected patients [48] , one of whom with a poor outcome (died) that had elevated levels of il-17 and il-10 which promote the th2 immune response, while the other patient who overcame the infection had increased levels of ifn-α, ifn-γ, and il-12, which promote the th1 immune response [48] . in general, it is crucial to induce early and strong innate immune responses against mers-cov infection to save lives. our vaccine candidate was able to induce production of the key cytokines of activated t lymphocytes toward cd4+ th1 cells, which is also an indication of upregulation of the antigen-presenting pathway. the vaccine also did not trigger the production of il-4, which is involved in the th2 response, which may be referred to the counter effect of the produced ifn-γ in il-4 producing cells [41] . finally, it is important to clarify that the in vitro antigen stimulation was done using the specific 9-mer peptide "cysslildy," this peptide is located at position 437-445 within the conserved region of the s glycoprotein among all identified mers-cov strains. this was selected in a computerized simulation that showed that this sequence has the highest b cell antigenicity plot and has the ability to form the greatest number of interactions with mhc-class i alleles [33] . although this epitope was not able to increase ifn-γ production level after in vitro stimulation, it resulted in a significant increase in il-12 production and a significant decrease in il-4 production in the vaccinated group. in addition, this epitope was able to increase il-4 production after in vitro stimulation in ad-cmv group. however, these findings were not enough to prove the claim that this epitope maybe has a crucial role in mers-cov pathogenesis. hence, further studies (such as in vivo or knockout experiments) should be conducted to address the role of this epitope in mers-cov pathogenesis. overall, the adenoviral vector encoding the s1 subunit of the mers-cov genome is a promising vaccine candidate against mers-cov infection. it was clearly demonstrated that this recombinant vaccine is distributed in host tissues. in addition, this construct could induce production of a specific anti-s1 igg antibody response as well as the th1 immune response, which was evident from the increased levels of pro-inflammatory cytokines in the vaccinated animals. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution mers: emergence of a novel human coronavirus middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov--are we on the verge of a pandemic? middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in 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coronavirus (mers-cov) are potent interferon antagonists delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? this study was supported by the deanship of research at jordan university of science and technology (grant no. 275/2014). ma and mua designed the adenovirus vaccine for the mers-s1 vaccine. ma and mk designed the mice experiments. mua, ma, and mk took the different tissue samples from the mice. mua performed the molecular assays and the immunohistochemical staining. ma and mua prepared the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord-352527-eeyqh9nc authors: zhou, yusen; yang, yang; huang, jingwei; jiang, shibo; du, lanying title: advances in mers-cov vaccines and therapeutics based on the receptor-binding domain date: 2019-01-14 journal: viruses doi: 10.3390/v11010060 sha: doc_id: 352527 cord_uid: eeyqh9nc middle east respiratory syndrome (mers) coronavirus (mers-cov) is an infectious virus that was first reported in 2012. the mers-cov genome encodes four major structural proteins, among which the spike (s) protein has a key role in viral infection and pathogenesis. the receptor-binding domain (rbd) of the s protein contains a critical neutralizing domain and is an important target for development of mers vaccines and therapeutics. in this review, we describe the relevant features of the mers-cov s-protein rbd, summarize recent advances in the development of mers-cov rbd-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy. middle east respiratory syndrome (mers) coronavirus (cov) is an infectious virus that was first reported in june 2012 [1] . mers-cov may infect people of any age, but older age, underlying comorbidity (such as diabetes mellitus, renal disease, respiratory disease, heart disease, and hypertension), and delayed confirmation or late diagnosis are all factors that affect mers disease outcomes and mortality [2] [3] [4] [5] [6] [7] . sex could be a factor in mers epidemiology, as more males seem to be affected than females [8] [9] [10] . mers-cov infection of women during pregnancy has adverse outcomes, with fetal mortality of~27%; however, only a limited number of pediatric mers-cov infections occur [11] [12] [13] [14] . at the end of december 2018, 2,279 laboratory-confirmed mers infections were reported globally (in 27 countries), leading to 806 deaths, and a mortality of 35.3%. among these infections, 1,901 (83.4%) were reported in saudi arabia, with mortality in 732 individuals (38.5%) (http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html). the largest mers outbreak outside saudi arabia occurred in south korea in 2015, with 186 cases and 38 deaths [9, 15, 16] . the most recent mers cases were reported in 2018 in south korea, the united kingdom, and malaysia, in addition to saudi arabia, the united arab emirates, and oman (http://www.who.int/emergencies/mers-cov/en/). mers-cov is thought to have originated in bats [17] [18] [19] [20] . mers-like viruses have been isolated from bats that use (at lower efficiency) the same receptor for cell entry as the mers-cov isolated from humans [21] [22] [23] . dromedary camels are potential intermediates for long-term evolution of mers-cov and seasonal zoonotic transfer of virus to humans [24] [25] [26] [27] . antibodies specific to host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [71, 78] . the dpp4 receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to speciesspecific variations [79] [80] [81] . in addition to dpp4, mers-cov can bind to sialic acid via the s1 subunit of s protein, or utilize the membrane-associated 78 kda glucose-regulated protein (grp78) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [82, 83] . the structures of mers-cov rbd alone and complexed with dpp4 have been determined ( figure 2 ) [77, 84, 85] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [77] . a number of rbd residues are located at the dpp4-binding interface, and they have a critical role in rbd-dpp4 binding [77, 84, 85] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp4. notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp4 [86] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd events [87] . in configurations with one, two, or three rbds rotated out, rbd determinants are exposed at the apex of the rbd-dpp4 complex, and they are accessible for interaction with dpp4 ( figure 3 ) [87] . mers-cov s protein has an important role in viral pathogenesis, determining host tropism and entry into host cells [58, 70, 71] . the s protein contains an s1 subunit at the n terminus and an s2 subunit at the c terminus. the s1 subunit is composed of the n-terminal domain (ntd) and rbd [58, 72, 73] . the rbd has a key role in the mediation of binding of mers-cov to cells expressing dipeptidyl peptidase 4 (dpp4) receptor, enabling the virus to enter into target cells by fusing with cell membranes through the formation of a fusion core ( figure 1c ) [74] [75] [76] [77] . the s protein requires host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [71, 78] . the dpp4 receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to species-specific variations [79] [80] [81] . in addition to dpp4, mers-cov can bind to sialic acid via the s1 subunit of s protein, or utilize the membrane-associated 78 kda glucose-regulated protein (grp78) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [82, 83] . the structures of mers-cov rbd alone and complexed with dpp4 have been determined ( figure 2 ) [77, 84, 85] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [77] . a number of rbd residues are located at the dpp4-binding interface, and they have a critical role in rbd-dpp4 binding [77, 84, 85] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp4. notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp4 [86] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd the mers-cov rbd core is colored in blue, the rbm is colored in red, and dpp4 is colored in green. the rbm residues directly involved in dpp4 binding are shown as sticks. dpp4, dipeptidyl peptidase 4; rbd, receptor-binding domain; rbm, receptor-binding motif; s, spike protein. the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (ad5)-directed expression of human dpp4 (ad5/hdpp4), hdpp4-transgenic (hdpp4-tg) mice, and non-human primates (nhps) [88] [89] [90] [91] [92] [93] [94] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table 1 . the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (ad5)-directed expression of human dpp4 (ad5/hdpp4), hdpp4-transgenic (hdpp4-tg) mice, and non-human primates (nhps) [88] [89] [90] [91] [92] [93] [94] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table 1 . a soluble nanoparticle vaccine formed in escherichia coli by the rna-mediated folding of a rbd-ferritin (fr) hybrid elicits robust rbd-specific antibody and cellular immune responses in mice, producing antisera that effectively block the binding of rbd to hdpp4 in vitro [89] . the adjuvants alum and the squalene-based mf59 significantly augment the antibody titers and t-cell responses induced by rbd-fr nanoparticle vaccines engineered with or without a ssg linker [89] . similarly, a chimeric, spherical vlp (svlp) vaccine expressing mers-cov rbd induces specific antibody and cellular immune responses in mice, preventing pseudotyped mers-cov entry into susceptible cells [90] . the protective efficacy of these two types of mers vaccine does not yet seem to have been investigated in a viral-challenge animal model. recombinant vaccines involving rbd subunits have been extensively studied for protection against mers-cov infection in mers-cov-susceptible animal models [93, [95] [96] [97] 100, 101] . a recombinant rbd (rrbd) fragment (residues 367-606) expressed in insect cells elicits an antibody response and the production of neutralizing antibodies in mice and nhps [88, 91] . it gives incomplete protection in mers-cov-challenged nhps, with the alleviation of pneumonia and clinical manifestations, as well as the reduction of viral load in lung, trachea, and oropharyngeal swabs [91] . a mers-cov s-protein rbd fragment containing residues 377-588 has been identified as a critical neutralizing domain [95] . a treatment regimen involving two doses of a fusion of this fragment and the fc region of human igg (s377-588-fc) four weeks apart is able to induce strong, long-term antibody responses (including production of neutralizing antibodies) in mice [98] . these responses are significantly greater than those with a single dose or two doses at intervals of one, two, or three weeks [98] . rrbds with single or multiple mutations corresponding to s-protein sequences of mers-cov strains isolated from humans or camels from 2012 to 2015 have also been studied [100] . all these rrbds bind rbd-specific neutralizing monoclonal antibodies (mabs) and dpp4, and are highly immunogenic, eliciting the production of s1-specific antibodies in mice, which cross-neutralizes multiple mers pseudoviruses and live mers-cov [100] . a trimeric rbd-fd protein formed by fusing a mers-cov rbd fragment (residues 377-588) to the foldon trimerization motif, binds strongly to dpp4, and elicits robust and long-term responses with the production of mers-cov s1-specific antibodies and neutralizing antibodies in mice, and protects hdpp4-tg mice against mers-cov infection [94] . the protection provided by existing subunit vaccines based on wild-type mers-cov rbd is not complete, with survival rates in hdpp4-tg mice after a mers-cov challenge of~67% for s377-588-fc and 83% for rbd-fd [94, 98] . however, a variant rbd (t579n) vaccine produced by masking a non-neutralizing epitope at residue 579 with a glycan probe has both functionality in binding dpp4, and antigenicity in binding four potent mers-cov rbd-specific neutralizing mabs (hhs-1, m336, m337, and m338) [93] . the t579n vaccine has significantly greater efficacy than the wild-type rbd vaccine, and it fully protects against a lethal mers-cov challenge in immunized hdpp4-tg mice [93] , demonstrating the possibility of developing rbd-based mers-cov vaccines with high efficacy. [102] [103] [104] [105] [106] [107] [108] [109] . these antibodies generally have greater neutralizing activity against mers-cov infection than non-rbd s1-based or s2-based antibodies [58, 103, 110, 111] . the prophylactic and therapeutic efficacies of rbd-targeting antibodies have been tested in ad5/hdpp4 mice, hdpp4-tg mice, and nhps [102, 104, [112] [113] [114] . in an earlier review, we described the antiviral mechanisms, in vivo protection, and crystal structures of previously reported mers-cov rbd-specific mabs, including mouse mabs mersmab1, 2e6, 4c2, f11, and d12, and human mabs lca60, mers-4, mers-27, regn3048, regn3051, 1e9, 1f8, 3a1, 3b11, 3b12, 3b11-n, 3c12, m14d3, m336, m337, m338, hms-1, and 4c2h [58] . in this review, we focus on newly reported antibodies targeting mers-cov s-protein rbd, or on newly identified features of existing mabs that were not described previously (table 2 ) [102, [112] [113] [114] [115] . rbd-targeting human mabs have been extensively reported. most of these mabs can neutralize pseudotyped or live mers-cov in vitro, and some have shown protection against mers-cov infection in animal models in vivo [102, [112] [113] [114] [115] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure 4) [102, [112] [113] [114] [115] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp4 (as is the case for gd-27, mca1, and cdc2-c2), or with binding to the rbd residues outside of the dpp4-binding interface (as seen with mers-4) ( table 2) . infection in animal models in vivo [102, [112] [113] [114] [115] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure 4) [102, [112] [113] [114] [115] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp4 (as is the case for gd-27, mca1, and cdc2-c2), or with binding to the rbd residues outside of the dpp4-binding interface (as seen with mers-4) ( table 2) . the human mabs mers-gd27 and mers-gd33 each recognize distinct regions of the rbd [113] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic50) for their use in combination than separately [113] . an analysis of crystal structures has indicated that mers-gd27 binds rbd at the dpp4-binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m336 [106, 113] . the mers-gd27 mab protects hdpp4-tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day 5 postchallenge, and higher survival rates (60% for pre-challenge vaccination and 40% for post-challenge vaccination) relative to control mice treated with an irrelevant mab [112] . the human mab mca1 was isolated from a mers survivor via the construction of a phagedisplay antibody library from peripheral b cells [114] . crystal structure analysis indicates that mca1 binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp4 ( figure 4a ) [114] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced the human mabs mers-gd27 and mers-gd33 each recognize distinct regions of the rbd [113] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic 50 ) for their use in combination than separately [113] . an analysis of crystal structures has indicated that mers-gd27 binds rbd at the dpp4-binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m336 [106, 113] . the mers-gd27 mab protects hdpp4-tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day 5 post-challenge, and higher survival rates (60% for pre-challenge vaccination and 40% for post-challenge vaccination) relative to control mice treated with an irrelevant mab [112] . the human mab mca1 was isolated from a mers survivor via the construction of a phage-display antibody library from peripheral b cells [114] . crystal structure analysis indicates that mca1 binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp4 ( figure 4a ) [114] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced lung disease, compared with unvaccinated controls, and undetectable virus titers 3 days post-challenge [114] . a probe-based single-b-cell cloning strategy has been used for the isolation of cdc2-c2 and cdc2-c5 mabs from a patient convalescing from mers, as well as for the isolation of jc57-11 and jc57-14 mabs from nhps immunized with mers-cov full-length s dna and protein [102] . all these antibodies have neutralizing activities against both pseudotyped and live mers-cov. among them, cdc2-c2 is the most potent against 10 pseudotyped mers-cov strains, with neutralization ic 50 values ranging from 0.002 âµg/ml to 0.011 âµg/ml [102] . crystal-structure analysis of the cdc2-c2 and jc57-14 fab-rbd complexes indicates that both mabs bind rbd in the "out" (exposed) position, with the cdc2-c2 rbd binding overlapping with the dpp4-contacting residues ( figure 4b ,c) [102] . in addition, cdc2-c2 prophylactically protects hdpp4-tg mice from mers-cov infection, resulting in no detectable viral replication in the lungs three days post-challenge, and no fatalities over 28 days of observation [102] . the human mab mers-4 also neutralizes pseudotyped mers-cov and, notably, displays synergistic neutralization in combination with the mers-cov s-protein rbd-targeting mers-27 and m336 mabs [106, 118] , as well as the s-protein ntd-targeting 5f9 mab, in each case with dramatic reduction of the ic 50 compared with individual mabs [115] . structural analysis of a mers-4-fab-rbd complex revealed that mers-4 binds the rbd from outside the dpp4-binding interface, rather than competing with dpp4 ( figure 4d ). unlike mers-27, which binds rbd regardless of its conformational state within the s trimer, mers-4 binds rbd in the "standing" position where its epitopes are readily exposed and accessible [115] . thus, mers-4 displays unique epitope specificity, and an unusual mechanism of action involving indirect interference with dpp4 binding through conformational changes, which may explain the observation of synergistic neutralization in combination with other mabs [115] . single-domain antibody fragments (vhhs), or nanobodies, are the antigen-recognition regions of camelid heavy-chain-only antibodies (hcabs), which do not contain light chains. vhhs are easily expressed with high yield, and they have intrinsic stability, strong binding affinity, and specificity to target antigens, and they have therefore been developed as important therapeutic tools against viral infection, including that of mers-cov [116, 117, [119] [120] [121] [122] [123] . four vhhs (vhh-1, vhh-4, vhh-83, and vhh-101) have been identified from bone marrow cells of dromedary camels immunized with modified vaccinia virus (mva) expressing mers-cov s protein, and challenged with mers-cov [116] . these vhhs bind mers-cov s protein with low k d values (0.1-1 nm), recognize an epitope at residue d539 of rbd, and neutralize mers-cov (prnt 50 , 0.0014-0.012 âµg/ml) [116] . these four monomeric vhhs have each been fused with a c-terminal human igg2 tag to generate four hcabs (hcab-1, hcab-4, hcab-83, and hcab-101), with a higher binding affinity and a longer half-life than the free vhhs [116] . studies of protective efficacy show that hdpp4-tg mice (k18) injected with monomeric vhh-83 (20 or 200 âµg per mouse) lose weight, and die within seven days post-infection, possibly because of the short half-life of the vhh. however, when the mice are injected with hcab-83 (200 âµg per mouse), which has an extended half-life (~4.5 days), protection against mers-cov is complete, with no viral titers or pathological changes in the lungs of virally challenged mice [116] . by immunizing llamas with a recombinant rbd fragment (residues 377-588) fused to a c-terminal human igg fc tag (s377-588-fc), we constructed a vhh library, and we used it to generate a monomeric vhh, nbms10, and a human fc-fused vhh, nbms10-fc [117] . both vhhs can be expressed in a yeast expression system to high purity, and bind rbd with high affinity, recognizing a conformational epitope (residue 539) at the rbd-dpp4 interface, and blocking the binding of rbd to dpp4. these vhhs, particularly nbms10-fc, potently cross-neutralize pseudotyped mers-cov strains isolated from different countries, hosts, and time periods [117] . importantly, the fc-fused nbms10-fc significantly improves the serum half-life of nbms10, and a single-dose treatment of hdpp4-tg mice with this agent completely protects them against lethal mers-cov challenge [117] . these single-domain vhhs demonstrate the feasibility of developing cost-effective, potent, and broad-spectrum therapeutic antibodies against mers-cov infection. compared with vaccines based on mers-cov full-length s protein, which have the potential to attenuate neutralizing activity or enhance immune pathology, vaccines developed from mers-cov s-protein rbd are safer, and they do not cause immunological toxicity or eosinophilic immune enhancement [55, 99, 110, 124] . moreover, rbd-based therapeutic antibodies are generally more potent than non-rbd s1-based or s2-based antibodies [58, 104, 111] . hence, rbd-based vaccines and therapeutic antibodies have the potential for further development as effective tools to prevent and treat mers-cov infection. despite their acknowledged advantages, there are some issues associated with rbd-based interventions that need to be addressed. for example, rbd is under a high level of pressure of positive selection, and mutations occur in the rbd-dpp4 binding interface that might reduce the efficacy of these treatments [100, [125] [126] [127] . one possible way to avoid this effect, and to delay the emergence of escape mutants is to combine rbd-targeting therapeutics with those targeting other regions of the s protein, or to combine antibodies recognizing distinct epitopes within the rbd [102, 128] . such combinatorial strategies could also dramatically reduce antibody neutralization doses, providing feasible means to combat the continual threat of mers-cov. some recent advances have been made in the structure-guided design of anti-mers-cov interventions. structurally designed inhibitors of the 3cl protease have demonstrated potency against mers-cov [129] . also, a structurally designed s-protein trimer in the optimal prefusion conformation is shown to elicit production of high titers of anti-mers-cov neutralizing antibodies [87] . indeed, based on the previous studies on the structural design of mers-cov rbd, non-neutralizing epitopes in the rbd can be masked, to refocus the immunogenicity of the rbd on the neutralizing epitopes, and thus to enhance its ability to confer immune protection [93] . results from these structure-based studies will help to inform the design of innovative rbd-based anti-mers-cov vaccines and therapeutics with improved efficacy. isolation of a novel coronavirus from a man 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spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization combining a fusion inhibitory peptide targeting the mers-cov s2 protein hr1 domain and a neutralizing antibody specific for the s1 protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this study was supported by the nsfc grant 81571983, and the nih grants r21ai128311, r01ai137472, and r01ai139092. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the authors declare no competing interests. key: cord-017615-zjr6csla authors: hillman, john r.; baydoun, elias title: food security in an insecure future date: 2016-11-25 journal: water, energy & food sustainability in the middle east doi: 10.1007/978-3-319-48920-9_12 sha: doc_id: 17615 cord_uid: zjr6csla food security in the middle east is directly affected by a challenging combination of ongoing destructive conflicts, a global economic downturn, widespread poverty, high population growth, corruption, intolerance, and the potentially damaging consequences of climate change. many arab countries demonstrate nearly all the features of those countries classified as poor, less developed, or failing to achieve the eight millennium goals. even the economies of the richer oil-exporting countries in the region have been seriously damaged by the downturn in oil and gas prices as new sources come on stream elsewhere and demand falls as a result of renewable sources of energy becoming available. in a previous article , we considered definitions of food security in the modern era of rising global populations, discussing how food security might be attained in terms of security of water and fossil-fuel-derived energy supplies, climate change, rapid urbanisation, changing dietary trends, and modification of the natural environment leading to depleted natural resources, increasing environmental pollution, and the need to introduce modern technologies. the concepts of sustainable agriculture and uncertainty were also addressed, notably in respect of fresh thinking about key components of agricultural systems. these included (babu and blom 2014) vertical and horizontal integration of farming-related businesses to allow adequate capitalisation for enhanced efficiency measures; (bardshaw and brook 2014) policy shifts to remove market-distorting subsidies, tariffs, import and export bans, and excessive bureaucracy; improved crop and livestock breeding, including entirely new species; (bolukbasi et al. 2015) automation in agriculture and horticulture; (breisinger et al. 2014 ) protected cropping; (cong et al. 2013 ) new-generation agrochemicals; (elasha 2010) new agronomic practices; (fan et al. 2014 ) novel foodstuffs; (fao 2011) habitat reconstruction and land renovation; (fao 2015) biofuels and biodiesel; (garland et al. 2014 ) periurban and urban agriculture; (grebner et al. 2014 ) industrial biotechnology; (grivetti and ogle 2000) farming the seas and oceans; long-term carbon storage; and (hillman and baydoun 2015) new ways of thinking about carbon trading. more recently, we reviewed mitigation and adaptation processes and strategies to address the impacts of climate change on food, water, and energy security in the arab middle east (hillman and baydoun 2015) . here, we consider potential adaptations to an insecure global future generally, and to the concerns in the arab middle east specifically, in the light of the economic realities of wide disparities in wealth, competition for resources, and widespread poverty in many parts of the globe, coupled to a relatively high population growth, on-going conflicts, attempted cultural genocides, potential conflicts, endemic corruption and nepotism, and epidemics of infectious diseases. most arab countries are classified as poor, less developed, or failing to achieve the eight millennium goals of the united nations, and these arab countries share several undesirable features (table 1) . even the much richer oil-exporting arab nations are under pressure. after a decade of relatively high oil prices, these nations have accumulated more $2.5 trillion in sovereign assets reinforced by substantial infrastructural investments supplemented by high levels of spending on imported military hardware. now, however, oil prices are under pressure as global oil and gas prices slumped in 2014 and remain depressed, possibly for the medium to long term as new sources of oil and gas come from hydraulic fracturing (fracking) and from iranian exports as the economic blockade on iran is being relaxed, coupled to greater energy efficiency in industry and new sources of renewable energy. this price depression has exposed the degree to which the economies of these arab oil exporters are dependent on oil and their failure in most instances to diversify their economies as their populations continue to expand alongside public expectations of continuing governmental largesse. nearly all of the immediate adaptations arab countries must undertake in order to adjust to a raft of severe insecurity issues require strategic planning and value-for-money infrastructural and civil-society improvements, and any preparatory changes in rural and urban areas will differ in scale and design. longer-term adaptations will be reliant on more stable conditions and a stepwise improvement of educational standards and attitudes. at the time of writing, no arab country is deemed to have acceptable levels of budget transparency according to the latest 2015 open budget index released by the international budget partnership (see www.internationalbudget.org). the future is especially insecure because of the persistence in the region of a combination of incompatible political and economic ideologies, religious and ethnic groupings overtly intolerant of others, introvert nationalism and disrespect of others, disconnection from democratic principles, profound cultural divides, ignorance -some wanton, inability to adapt to modernity, and malevolent community and national leaders. from a noble history of toleration, hospitality, and learning, arab society is fragmenting, defiled by the actions of relatively few. arabs are killing arabs either directly or indirectly; arabs are inflicting as-yetuntold horrific crimes on other arabs either directly or indirectly. attacks on arab countries by their neighbours might be used as an excuse to divert attention from their own failing donor-dependent economies or social structures, or most often to steal resources and ensure that the neighbouring country is suppressed from developing normally. at the global level, many would say that future conflicts and insecurity in much of the world are inevitable, simply because of the impacts of expanding global populations and the obvious competition for limited resources. the intensity of this competition must be analysed in context of the alarming table 1 fourteen features of countries classified as poor, less developed, or failing to achieve the eight united nations millennium goals. the listed features are closely interrelated 1. poverty common in both the urban and especially the rural poor, poverty is sometimes concentrated in regions, often in marginalised ethnic or religious groups, and may relate primarily to girls and women. many of the urban poor operate in the unofficial economy. the rural poor tend to be land-constrained, dependent on rain-fed, low-yield subsistence agriculture with little or no access to modern technology (modern cultivars and livestock breeds, fertilisers, pesticides, automation, agronomy advice, veterinary support), and usually do not own their land. the rural poor encounter barriers to trade (e.g. transport, storage) and are unable to meet quality assurance standards. with no or limited access to social benefits (primarily pensions, child support, education, training, and healthcare), the poor may be hungry, thirsty, suffer ill health and low standards of accommodation, and die early. the poor are susceptible to exploitation. 2. hunger and thirst access to food and potable water may involve substantial travel on foot, and the basic requirements may only be met wholly or in part by humanitarian assistance. food quality and safety are usually low, and cooking is often dependent on wood for fuel. symptoms of malnutrition are prevalent. 3. disease slum dwellings, insanitary conditions, poverty, and hunger lead to a vulnerability to pandemics, made worse by poor or no public health provision. high maternal and child mortality and low general life expectancy characterise poverty and there is a reliance on traditional and/or herbal medicine. crops and livestock are subject to catastrophic attacks by pests and diseases. 4. poor environmental management poor countries suffer a depletion of their natural resources, including freshwater supplies and native flora and fauna. mineral and fossil-fuel resources are extracted to be exploited by industries in other more-developed countries. land, water, and the atmosphere may be polluted with few or no remediation efforts. national, regional, and international environmental regulations are not properly implemented. agricultural soils tend to be subject to erosion, salination, solarisation, desertification, and nutrient depletion. even without climate-change predictions, the general anthropogenic environmental degradation currently taking place increases the vulnerability to flooding, sand storms, ill health, and displacement of peoples. most poor countries are enduring adverse climate trends, and climate-change predictions point to even harsher conditions (erratic rainfall with rising temperatures, heat waves, hot extremes, and storms; rising sea levels and acidification of seas and oceans; increased desertification with effects on agriculture as well as the natural flora and fauna; socio-economic and health implications). 5. poor infrastructure quantity and quality of the built infrastructure tends to be low, or even absent (e.g. roads, ports, airports, telecommunications and access to the internet, hospitals and clinics, reliable power and fuel supplies, potable water sources, sanitation systems and sewage disposal, protected natural environments, cold and pest-free storage of agricultural and horticultural produce). the cost of living is worsened by relatively high overland transport costs. facilities may be inadequate to meet demand and maintenance neglected. rapid urbanisation and conflicts exacerbate infrastructural deficiencies. 6. corruption political autocracy coupled to a lack of transparency in government and public services, a weak judiciary, lack of consultation on major issues, and suppressed media enable a climate of corruption and nepotism to permeate all areas of society, including schools, colleges, and universities. a low regard for human rights, democratic processes, health and safety measures, and international law, is made worse by an overburdensome bureaucracy (staffed by poorly paid civil servants and police) dependent on bribery to function. corruption allows laws to be broken with impunity, and protests ignored. in most areas of society, a lack of altruistic leadership and (continued) poor countries manufacture few value-added products, offer little or no advanced training, and lack participation in the global knowledge economy. there is little or no foreign direct investment and free trade is constrained. financial assistance from donor countries is increasingly being audited to ensure compliance with attempts to prevent unauthorised expenditure. many poor countries have not managed to have constructive relationships with potential donor countries, and some try to align themselves with one of the main international political power blocs. unrelieved debt burdens have led to high interest rates on loans and difficulty of obtaining credit. private savings are minimal and the country may be subject to periodic flights of capital. their economies are grossly imbalanced with little spent on healthcare, education, and other social benefits compared with defence and vanity projects. the economy may be damaged by previous and/or ongoing conflicts, and may in any case have limited absorptive capacity properly to manage additional resource inflows and outflows. agriculture, unofficial transactions, and remittances from those working abroad contribute disproportionately to the real economy. 8. poor education limited or no access to free schooling accounts for a general low level of literacy and numeracy, especially of females. this is reflected in the absence of high-grade internationally competitive universities, colleges, and research institutes, despite a high level of parental financial sacrifice to secure a supposedly good education for their children. science and technology tend to be poorly taught and there is undue influence of religions with regressive attitudes to modernity. a lack of investment in research and development (r&d) and a lack of a critical mass of scientists, engineers, and technologists impede industrial development, and prevent those that remain from joining international consortia, participate in learned societies, access essential literature and training programmes, and have the opportunity to use state-of-the-art instrumentation, software, and laboratory consumables. the best educated and the most talented and entrepreneurial usually emigrate, leading to a brain drain. intellectual property rights are often ignored and little benefit is derived from traditional knowledge and its products tend to be exploited in other countries. crucial demands and needs of less-developed countries are rarely the targets of major international r&d projects. 9. gender inequality low educational attainment in most girls and women is reflected in female political, economic, and social representation and participation failing to match their proportion of the general population. cultural and religious influences often lead to females being regarded as inferior to males. family planning is limited, and in the absence of social security and reasonable incomes, large families are the norm. 10. high population growth this is a feature of the poorest social groups, and correlates with low life expectancy and gender inequality. in some countries high population growth may exceed the economic capacity of the country to feed itself, leading to a propensity to generate refugees, displaced persons, and terrorists. 11. vulnerability to transnational terrorism terrorism often relates to a combination of one or more of the following: poverty, hunger, poor educational attainment, disconnection from democratic principles, susceptibility to indoctrination by intolerant religious ideologies, criminal activities, and psychiatric disorders. poor countries are usually unable to defend themselves from terrorists, and groups of terrorists may receive covert support from other countries and agencies. poor countries may form the battlefield for fighting between different groups of terrorists. recovery from terrorism and conflicts in general may take several generations, and lead to psychological and physiological after-effects in the survivors and their progeny. projected changes and options for adaptation as well as mitigation (curbing emissions) detailed in the latest 2015 fifth assessment report of the international panel on climate change (ipcc, www.ipcc.ch/). the immediacy of food security during social instability in the arab region forms the backdrop to this article, rather than the longer-term infrastructural and social transformations needed to mitigate and adapt to climate-changing emissions, transformations that demand political stability and sophistication. the global population is estimated to be around 7.2 billion at present and there is an 80% probability that it will increase to between 9.6 billion and 12.3 billion in 2100 (garland et al. 2014) , so stabilisation of the population is highly unlikely this century. moreover, human population reduction is not a quick fix for environmental problems, and even a catastrophic event that killed billions of people would have relatively little effect on the overall impact of humans on the environment 12. weak public sector low salaries, complex and inefficient bureaucratic processes, poor educational attainment, incompetence, widespread acceptance of corruption through bribery and political interference, and the lack of an investigative free media, account for the justifiable lack of confidence in the public sector. a low regard for human rights, legal processes, and justice undermines societal advancement. there is also poor custodianship of cultural heritage and essential infrastructure. 13. neighbouring countries many poor countries have reliance on and vulnerability to neighbouring countries for access to water, transport and communication networks, foodstuffs, energy, control of environmental issues (e.g. desertification, flooding, biodiversity protection, pollution etc.), and security. neighbouring countries may (a) create security problems by aiding terrorists and insurgents, (b) provide an uncontrolled source of refugees, (c) invade, (d) steal water or other natural resources, and/or (e) issue mendacious media releases or operate diplomatically to undermine the confidence of donor countries, aid agencies, and investors. the more-developed countries rarely understand the sheer difficulty of managing a poor country facing inter-ethnic conflict, and terrorism and poverty. 14. international agencies, non-government organisations, international media and the united nations poor countries are monitored by a plethora of international and national bodies, and extensive reports generated, but the necessary actions -political and military -to solve the main issues and problems are rarely carried out. unwarranted aggression inflicted on other countries -poor or not, or the suppression of their populations or specific parts of their populations by nation states eventually must be counteracted by military intervention. sadly, poor nations are oftentimes regarded as pawns in international power struggles, and remain either exploited for their resources, or ignored. moreover, economic downturns in donor countries reduce the level of aid and absorption of refugees, made worse by certain countries cynically failing to meet their initially well-publicised aid pledges. (bardshaw and brook 2014) . population increases could undermine attempts to ameliorate attempts to reduce climate-modifying emissions. climate-change predictions for the arab region are deeply concerning. most scientists support the conclusions of the latest fifth assessment report of the ipcc. formed in 1998 by the united nations environment programme (unep) and the world meteorological organization (wmo), and relating closely to the un framework convention on climate change -the main multilateral forum for addressing climate change, ar5 comprises three working group reports and a synthesis report with its summary for policymakers. the main issues are covered in detail: observed changes and their causes; future climate changes and their risks and impacts; future pathways for adaptation, mitigation and sustainable development; and a more detailed analysis of adaptation and mitigation including policy options, technology and finance. in the arab human development report authored by balgis osman elasha, (2010), the impacts of climate-change projections in the arab region are given in stark detail. in the coming decades, arab societies and their industries will be profoundly and adversely affected by projected temperature increases in excess of 4 c and severely reduced rainfall, threats of increasing frequencies of impacts originating from el nino events, changes in the seasonal distribution and predictability of rainfall, depleted aquifers, reduced river flows, rising sea levels, flash floods, in addition to increased numbers of dust storms and hurricanes. as the arab region itself is a relatively small direct contributor to global greenhouse-gas emissions, although its fossil-fuel exports and its importation of goods that took energy to create are substantial contributors to them, adaptation must be a crucial factor in policy developments in more settled times that will themselves be dependent on stable and peaceful arab countries. questions arise as to whether organisations such as those overseen by the united nations, multilateral groups such as the european union, individual nations and several charities will be capable of initiating and then maintaining peaceful conditions and food security. food and other forms of aid are likely to face increasing demands at a time when the economies of many donor countries are enduring continuing austerity and recessionary conditions, and when there is growing but unjustified cynicism about the effectiveness of these aid organisations. irrespective of the many estimates that total global food production can readily meet the needs of the present global population, political reality is that all countries and people are patently not equal and are unlikely to be so for the foreseeable future. aid can assist in partially rebalancing the inequalities, not least where several countries in the arab region are currently in turmoil and others are deeply troubled. some regard most of the arab world as regressing, out of synchrony with, and lacking sympathy from, the world at large. food costs and poverty are primary concerns, exacerbated by insecurity of energy and water supplies as well as rampaging insurgents and those wishing to impose unacceptable regimes and suppression of minorities, denying their citizens proper democratic freedoms. wars and conflicts are all too easily incited in the absence of strong democratic civil society involvement and usually quickly bankrupt countries; destroy nearly all parts of their economies; ruin infrastructure including homes, businesses, transport and communication networks; disrupt family life and social interactions; generate displaced people and refugees; and attract foreign interference, including active participants. war crimes are commonplace. psychological aftereffects are noted in civilians and combatants long after fighting has died down. populations in the arab middle east have endured asymmetric warfare between countries with vastly different military capabilities, provoking guerrilla tactics, chemical warfare, civil war, and even unconventional warfare through acquiescence, capitulation, and clandestine support for long-term insurgencies. wherever they occur, warfare and conflicts are always associated with the participants having distorted views of history -some drawing on ancient history -with widely diverse concepts of ethics, and complicated by ethnic and profound religious differences. little consensus exists on triggering factors, theories, and outcomes. prodigious sums of money have been expended in the arab middle east on armaments and defence forces to the exclusion of adequate investments in social welfare, education, and research and development. political instability for whatever reason usually leads to food insecurity and conflict. during social disturbances and the onset of widespread conflict, the normal mechanisms underpinning food production, importation, storage, processing, transport and retailing are profoundly disrupted, creating conditions that promote the formation of ghettos, and further stimulate corruption, robbery, and the black market. normal policing and social order collapse, social behaviour degrades, and criminal activities dominate. as the economy collapses, the terms of trade are transformed. disease epidemics become manifest. the restoration of domestic and business normality takes years and may never be achieved within one or more generations. food insecurity itself may lead to political instability, not least in a world of global intercommunications when citizens of a poor country or region can view with understandable envy the lifestyles of those in rich countries or communities. it is therefore a basic duty of political and community leaders to ensure that food security is a foremost priority for those people within their sphere of responsibility. insecurity of food supplies can be created by adverse weather conditions; the depredations of pests, weeds, and diseases; and salt contamination that can be caused by poor irrigation and agronomic practices. around 62 million hectares of hitherto fertile land on earth have been damaged by salt. according to economics of salt-induced land degradation and restoration (qadir et al. 2014) , only tree planting, deep ploughing, and growing salt-tolerant crops coupled to digging drains and dykes around the affected area can address the problem. neglecting the health of africa's soils, many of which suffer almost irreversible degradation and nutrient deficiency, will lock the continent into cycles of food insecurity for generations to come, according to the 2014 montpellier panel report. indeed, 2015 was designated the 'international year of soils' by the 68th un general assembly. since the 1920s, there are particular issues relating to selection pressures on destructive pests, weeds, and diseases in the vast monocultural single-cultivar agricultural systems that are also the present-day main sources of global food aid. many of these main producing areas are experiencing irregular rainfall patterns and failing irrigation arrangements. crop failures in these areas have quick knock-on effects on volatility of the global agricultural commodity markets; this is a situation likely to get worse as the population inexorably increases and demands more food. moreover, the world has yet to experience the sort of dramatic harvest failures that occurred in the 1930s and before. another aspect of the agriculture sector (generally accepted to include crops, livestock, fishing, and forestry) in the developing world is that it absorbs circa 22% of the economic impacts caused by medium-and large-scale natural hazards and disasters. between 2003 and 2013, these events in developing countries affected more than 1.9 billion people and caused more than $494 billion, but agriculture only received about 3.4% of all humanitarian aid (fao 2015 www.fao.org/emergencies/how-we-work/resilience/en/). delegates from fao at the conference announced the launch of a facility that will focus on bringing together technical expertise and financial resources with the aim of building greater resilience of agriculture to natural extreme weather events. the combination of advanced crop and livestock breeding, agrochemicals, automation, better agronomy and livestock husbandry, increases in the land area farmed, and efficient larger-scale better-capitalised production units have collectively prevented malthusian disasters. since 1950, food production has more than quadrupled, using less than 1% more cultivated land, allowing civilisation to proceed and expand. food security is no longer an issue in many countries, and the global economy, human health, and societal development have been, for the most part, positively influenced by agricultural advancement. in the period 1900-2010, when the global population increased from around 1.5 billion to around 7 billion, average agricultural commodity prices decreased by an average of 0.9% p.a. because supplies rose faster than demand (unpublished presentation by prof. ingo pies in 2014 to biennial development meeting of pottinger). nonetheless, feeding any substantial increases in the global population will only be possible by technological innovations because of the ecological limitations on increased water and fertilizer supplies and increasing the area farmed. likewise, various climatechange predictions amplify justifiable concerns about global agricultural productivity. supply and demand market dynamics are complex and ultimately resilient to political interference, although many countries and trading blocs try to manipulate production by tariffs, export bans, subsidies, inhibiting technological developments and market processes. the spikes in prices of traded wheat, rice, maize, and soya in 2008 and 2011 were initially blamed on speculation, especially the index tracking funds and derivatives markets. yet this type of speculation does not trade physical goods but price risks, and is therefore a form of insurance market. in addition, speculation would be expected to be associated with high stocks as farmers opt for storage rather than sales. in 2008 and 2011, however, stocks were very low and caused the price rises. even so, government policy failures, including protectionist export bans and inadequate promotion of agricultural efficiency, contributed to panic buying, as exporters reduced their offer and importers increased their demand in response. calls by civil society groups to ban speculation by index-tracking funds and derivatives markets were always and continue to be profoundly misguided. all countries should have policies to sustain and constantly review food production and supply, especially if there is a significant dependence on food imports. reasonably substantial reserve food stocks are essential cushions to prevent price bubbles and food shortages, but excessive stocks can distort markets such as when they are released in large quantities as general food aid and undermine the operation of normal agriculture markets in developing countries. some price volatility is an essential component of healthy competitive markets, driving adaptation, risk taking and innovation. debates about the environmental costs of different kinds of agriculture, not least in terms of water and energy security in respect of the arab middle east, and in terms of the destruction of natural habitats and loss of biodiversity, as well as cultural and other changes, have stimulated possible strategies to address these concerns. one approach is for each country to have a "roadmap" for its agricultural development, and these roadmaps might be aggregated into a regional roadmap. the us report: a science roadmap for agriculture -cited as task force on building a science roadmap for agriculture, national association of state universities and land-grant colleges, experiment station committee on organisation and policy, "a science roadmap for the future", november 2001 (www.nasulgc.org/comm_food. htm) http://agsci.oregonstate.edu/files/main/roadmap2.pdf pioneered a way to define the needs of agriculture and help shape the future direction of the various strands of agriculturally relevant science. this impressive us-specific study followed a conceptual framework of needs to (a) be competitive in a global economy; (b) add value in future harvests; (c) adjust agriculture to a changing climate; (d) be good stewards of the natural environment and natural resources; (e) make agricultural enterprises profitable; (f) make families and communities strong; and (g) modify foods for improved health and safety. one important relevant outcome of this work is the obvious requirement to grow crops and practice livestock husbandry in the most appropriate environments, enabling different environmental zones to have a critical mass of expertise and facilities. for the water-constrained arab middle east, this would mean increased reliance on food imports that could also be regarded as a form of water importation. but this would be possible only if there were formal agreements between food-producing and food-recipient countries, and these agreements were economically and politically stress-resistant. to this caveat would be questions of how to pay for imports, and acquiring resources required to redirect the agricultural workforce into other wealth-creating activities. introduction of a logical, science-led agricultural roadmap in the region may be impossible at present but needs to be initiated. on a global level, food commodity and non-food agricultural commodities prices have declined by 17-18% in the period mid-september 2014 to mid-september 2015 (see the economist commodity-price index (2015) in www.economist.com/indicators), reflecting relatively clement weather conditions in most of the producing areas, greater production efficiency, balance sheets strong enough to bear losses, competition to gain market share, and continuing investments. importing countries, however, are affected by the strength of their currencies amongst other factors, such as social upheaval. unlike other commodities whose prices direct reflect industrial demand, prices of foodstuffs reflect the effects of weather, pests and diseases, the demands of a rising global population, and many kinds cannot be readily stockpiled. a further factor operating at the global level is the domination of farm supplies by six international companies: monsanto (the largest seed producer), syngenta (the largest agrochemical producer), bayer, basf, dow chemical, and du pont). all of these companies have been active in acquiring other companies and patents, thus reducing competition, and potentially reducing innovation as they robustly defend their intellectual property. in this era of low commodity prices and developing resistance to older-type herbicides, farmers are constrained by input costs of fertilisers, seeds, agrochemicals and veterinary medicines that have steadily risen in over the past decade, only partially alleviated by the recent reduction in fuel and lubricant costs. pandemics -epidemics of infectious or contagious disease that have spread through populations across a large region, crossing international boundariesdrastically curtail food production and distribution, aggravating poverty in both the rural and urban poor. pandemic-causing diseases include the ever-present cholera, influenza such as the 1918 and 2009 h1n1 outbreaks, typhus, smallpox, measles, tuberculosis, plague (yersinia pestis), leprosy, malaria, human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids), viral haemorrhagic fevers (ebola, marburg, crimean-congo, lassa, rift valley, dengue, yellow fever, etc.), and now there are new diseases such as severe acute respiratory syndrome (sars). vaccine development is necessarily slow, and treatment of bacterial diseases is hampered by the rapid development of antibiotic resistance. the propensity of diseases to mutate, acquire new vectoring capabilities, have reservoirs in wild animals, and even persist in spore form, mean that there must be constant vigilance. as one of the major global food-producing bloc of nations, and as one of the major food importers and donors of food and other forms of humanitarian aid, the european union (eu) bears crucial responsibility for the deleterious effects of its complex and highly bureaucratic common agricultural policy. its massive subsidy regimes impact adversely on global markets and its well-meaning but often poorly thought-through environmental regulatory decisions are not based on sound scientific evidence. so-called "greening" policies are being introduced that may be deemed to enhance the environment but are likely to decrease profitable production. social measures to support small-scale inefficient producers also distort the global marketplace. likewise, the series of restrictions being introduced on the use of a wide range of agrochemicals within the eu and for imported commodities, without carrying out proper impact assessments and fast-tracking alternatives, imperil production. in the medium to long term, a more serious issue is the virtual ban in genetically modified (gm)) crops, inhibiting their uptake in countries intending to export to the eu as well as suppressing state-of-the-art research and development and associated investments in eu countries. of particular relevance in this regard is the recent and largest statistically rigorous review of the agronomic and economic effects of the current range of commercially available gm crops on farming (klã¼mper and qaim 2014) . in examining publications between 1995 and march 2014, it is therefore a near-complete survey. in essence, the two main types of gm crop -resistance to insect pests and tolerance to the wide-spectrum weedkiller glyphosate -conferred considerable yield improvements and much higher profits than conventional crops. gm crops and related products in the development pipeline were not considered, and these promise great advances in nutrition enhancement, environmental clean-up, new medicines, new products for manufacturing industries, and improved crop and forest species. moreover, existing gm crops have greater impacts in poorer countries than in richer countries because their insect pests and pernicious weeds are more difficult to control. by including non-peer-reviewed papers (book chapters, working papers, conference papers etc.) as well as peer-reviewed papers for the meta-analysis, it was possible both to correct for academic bias in focusing just on the most dramatic effects, and include data for many ancillary effects, such as the effects of fertilisers. the eu ban on gm crops is therefore denying poor exporting countries from reaping the full benefits in yield, profitability, and commodity quality, in addition to reducing potential eu food-aid exports. one positive feature of the eu is the bureaucratic system to improve and monitor the quality and safety of foodstuffs, from raw ingredients through to ready-meals, and gm crops have been found to be safe. that for many years most of the feed protein in the eu comprises imported gm maize and soya bean, the issue of gm crops should not be ignored for much longer. opposition to the processes of modern genetic modification and ownership of the processes (often deeming them to be "unnatural"), and disregarding the quality, safety, and valuefor-money of the product, actually condemns conventional agricultural practice, and demonstrates ignorance of naturally occurring mutations and horizontal gene transfer. even so, in early november 2014, the newly elected president of the european commission, j-p juncker, in what is widely regarded as a blatant act of appeasement to greenpeace and other so-called "environment" pressure groups, sacked the eu's chief scientific advisor, professor anne glover, for her support of gm technology, compounding this regressive stance with abolition of the post. all countries and trading blocs should have influential and competent teams of chief scientific advisors. research and development in gene identification, construction, insertion, editing, and expression, coupled to high-throughput phenotyping are collectively revolutionising agricultural, horticultural, and forestry sciences. gm technology is not simply the insertion of genes using various technologies from a similar or different species into a recipient organism. it includes the concept of gene silencing -the prevention the reduction of expression of certain genes -a process that can take place at either the transcription or translation cellular processes. it is not the equivalent of gene knockout but is essentially gene knockdown because the methods to silence genes do not completely eliminate the expression of a specified gene. the methods to silence genes include rna interference (rnai or posttranscriptional gene silencing), small interfering double-stranded rna (sirna), and crispr. of special interest is the crispr (clustered, regularly interspaced, short palindromic repeats) toolkit that is derived from research on prokaryotic antiviral systems and currently involving the cas9 and cpr1 endonucleases (jinek et al. 2012; cong et al. 2013; bolukbasi et al. 2015) . as viruses constantly evolve to escape from these antiviral systems, bacteria probably evolve new systems. crispr technology is able to recruit heterologous domains that can regulate endogenous gene expression as well as label specific genomic loci in cells, so that is feasible to engineer germ lines and thus the path of evolution. this technique is replacing methods using mutagenic agents, virus vectors, zinc-finger nucleases and transcription activator-like effector nucleases (talens). its relative simplicity and evolutionary significance for all life forms, including humans, means that internationally agreed regulatory frameworks are essential. the technology does not involve implanting genes from one organism into another, and is not therefore creating transgenic organisms; it is gene editing. there is now realistic expectation of new perennial cereals; incorporation of c-4 photosynthetic characteristics in existing c-3 crops; enhancement of nitrogen fixation by free-living soil microorganisms in the vicinity of crop roots; tolerance and resistance to biotic and abiotic stresses; and modification of lignification, texture and endogenous components (such as vitamin content, acrylamide in potatoes, antinutritionals, toxins, proteins, oils, and carbohydrates) of a wide range of existing and potential crop species (see www.isaaa.org/kc/cropbiotechupdate). besides the present-day generation of improved livestock species and new forms of husbandry, the use of balanced diets based on competitively priced synthetic amino-acid and fatty-acid products will lessen the need for large-scale soya and maize production. ancillary advances are taking place in mechanisation; diagnostics; predictive modelling and decision-support systems; remote sensing; protectedcropping systems; and weed, pest, and disease control. diminution of abiotic and biotic stresses in the field and under protective cropping is now the focus of major research initiatives. of the circa 250,000 species of angiosperms, according to the food and agriculture organisation of the united nations (fao 2004) , only 30 species provide 95% of human energy needs and only four species (rice, wheat, maize, and potato) account for 60% of energy intake, and 75% of crop diversity was lost in the last century. around 80% of human calorie intake comes from 12 crop species (grivetti and ogle 2000) and 50% comes from just three grasses -wheat, maize and rice (see www.knowledgebank.irri.org/ericeproduction/importanceofrice.htm) dependency on such a narrow genetic base is a threat to food security and is only partially alleviated by investments in in situ and ex situ plant gene/seed banks, germplasm collections, and dna libraries. just 7000 species of angiosperms and gymnosperms have been cultivated for human consumption in human history, with around 80,000 angiosperm species yet to be discovered. in theory, most angiosperms should be capable of being biotechnologically modified for food and non-food uses. will scientists in the arab middle east fully participate in these exciting developments? valuable collections in the arab region are inadequately respected for their worth under peaceful conditions but are now extremely vulnerable during this period of war-like conditions and enduring financial pressures. coming into force in 2004, the international treaty on plant genetic resources for food and agriculture (international seed treaty) was designed to complement the 1993 convention on biological diversity (cbd) and was designed to guarantee food security by (a) conservation, exchange, and sustainable use of all types of plant genetic resources; (b) offering fair and equitable benefit-sharing; and (c) recognition of farmers' rights. critics of the international seed treaty point to great variability across countries of access to collections and interpretation and implementation of farmers' rights. moreover, in adopting the cbd's outlawing of biopiracy -the uncompensated commercialisation and profiteering of seeds, propagules, growing plants, and their products from source areas -has severely inhibited the acquisition and exchange arrangements in collections until better processes come into force. gap analyses are methods to identify gaps in ex situ collections of wild-plant relatives of agriculturally relevant species as a means to guide efficient and effective collection strategies (villegas et al. 2010) . a gap analysis by the international center for tropical agriculture managed by the global crop diversity trust and the millennium seed bank in kew examined 29 priority gene pools of the globally most important food crops and their wild relatives. most at risk were eggplant, potato, apple, sunflower, carrot, sorghum, and finger millet (see www.cwrdiversity. org/conservation-gaps/). ongoing conflicts and disorder in the arab region justify an independent review of a regional red list index based on the list of threatened species released by the international union for the conservation of nature (www. iucn.org) in order to evaluate the extinction risk of species and subspecies of the natural flora and fauna. certain individual countries in the eu are important donors of humanitarian aid in their own right. according to a recent report (november 6, 2014) from the organisation for economic co-operation and development, economic stagnation especially in the eurozone portion of the eu poses a major risk to world growth. if the stagnation were to continue or even get worse, humanitarian assistance would inevitably be reduced, and countries that hitherto were willing donors would become increasingly introverted. as evidenced by growing problems of graft, corruption and authoritarian government in certain (but not all) members of the eu that were formerly dictatorships or in the sphere of influence of the former soviet union, democratic norms (human rights, respect for minorities, tolerance, free press, independent judiciary and rule of law, active civil-society groups, transparency of accounting for taxpayers' money etc.) take time to become bedded into the fabric of society. in considering the medium-to-long-term future of the eu, it is sobering to note that throughout european history, confederations between its diverse nations and subsets have rarely persisted unless full political, legal, monetary, and more profoundly, cultural fusion had taken place. all governments worthy of the title must ensure that there are relief mechanisms to enable the provision of basic food supplies and fresh water together with functioning standby electricity-generating equipment in unsettled times caused by natural or man-made disasters. surely governments have the ultimate responsibility to attend to the needs of their people and not themselves. rarely observed, governments need genuine food, nutrition and agricultural experts as an integral part of the decision-making hierarchy. such experts must have a proper understanding of the pre-conflict or pre-disaster food, fresh-water, and energy supplies and their distribution systems, and how they can be safeguarded, modified and employed to proper effect, and how alternative mechanisms can be deployed. sadly, this aspect seems to be neglected at the present time in the chaotic condition of certain countries in the middle east. much can be learned from countries in europe during the wars that raged in the nineteenth and twentieth centuries. simply standing by and watching the population adapt slowly to acquiring barely adequate water and food supplies inflicts untold misery on innocent people. all governments should have readily accessible emergency supplies (reserve stocks) and transport systems, and be willing to introduce rationing if need be. special protection measures are needed for water supplies and farms to make sure production can continue no matter the degree of impairment. in more settled times, each government should establish a group of experts to construct interactive databases as the foundation of an agriinformatics and metrics organisation. this would collate information on supply and demand changes, supply-chain details, imports, crop and livestock genetics, commodity production levels, labour-force composition, pricing, inputs, availability of decision-support systems, advisors and research bodies, grant funding, biotic and abiotic stress factors, natural resource constraints, predictive modelling of shocks to the agricultural system and disruptive events, etc. other research organisations would interact with this organisation to ensure best practice and enhance agricultural resilience, demonstrate efficient use of inputs, exploit wastes, optimise the use of mechanisation, and foster skills. arab countries still have to utilise fully the international capabilities and potential of (international centre for agricultural research in the dry areas -currently based in beirut given the conflicts in syria) and other members of the cgiar consortium (formerly the consultative group on international agricultural research). the plethora of aid agencies offering humanitarian and development aid encompass those that are organised by a single government, multilateral donors, non-governmental organisations, philanthropic and charitable organisations, businesses, and individuals. reliefweb (www.reliefweb.int) provides a relatively comprehensive directory of humanitarian organisations. fragmentation of the total aid effort is becoming a worrisome issue. the international committee of the red cross, part of the international red cross and red crescent movement along with international federation of red cross and red crescent societies and 189 national societies, is mandated internationally to uphold the four treaties and three additional protocols of the geneva conventions. these conventions are rules that apply in times of armed conflict both within and between countries, and define the rights of civil and military prisoners and protections for wounded people and for civilians. weapons of war are dealt with by the hague conventions and the biochemical warfare geneva protocol. enforcement of the conventions is through the un security council but is rarely invoked, primarily because of profound ideological differences about democracy and human rights between the five permanent members of the security council with veto powers, so there tends to be diplomatic reliance on regional treaties and national laws. parenthetically, there are ten non-permanent members of the security council without veto powers that are elected by the general assembly of the united nations for a two-year period. on 19 november 2014, the un security council pledged to counter the global terrorist threat and increase cooperation to address the perils posed by foreign terrorist fighters such as those that are a notable feature of conflicts in the arab region. the un office on drugs and crime is also involved in this initiative. other international related treaties include the united nations multilateral treaty referred to as the geneva protocol or convention relating to the status of refugees as well as the declaration on the protection of women and children in emergency and armed conflict adopted by the united nations in 1979. humanitarian aid is distinguished from humanitarian intervention, which involves armed forces protecting civilians from violence or genocide. the united nations office for the coordination of humanitarian affairs is mandated to coordinate humanitarian responses, usually in concert with the international committee of the red cross. valuable reference material can be found in (a) grebner et al. (2014) , the state of hunger in developing countries as a group has improved by 39% since 1990. even so, the level of hunger is still serious with an estimate of 805 million people continuing to go hungry. the highest levels are south of the sahara and south asia. in the ifpri global nutrition report 2014, evidence is summarised to show that improvements in nutrition status will make large contributions to sustainable development goals, namely poverty, food, health, education, gender, and employment investment in nutrition has a highest benefit ratio. projections from the world health organization (who) and unicef demonstrate that the world is not on track to meet any of the six world health assembly (wha) nutrition targets (reducing child stunting, reducing anaemia in women of reproductive age, reducing low birth weight, reducing the number of overweight children, increasing exclusive breast feeding, and reducing child wasting), although many countries are making good progress in meeting nutrition outcomes. the manifestation of malnutrition is changing as countries are now facing complex, overlapping, and connected malnutrition burdens. three of the chapters in the ifpri publication resilience for food and nutrition security (fan et al. 2014 ) are germane to this article. breisinger et al. (2014) briefly mention the arab spring and uses egypt, somalia, sudan, and yemen as case studies of conflict-affected countries. mabiso et al. (2014) have specific reference to the syrian refugee crisis, and take a global overview of the complex relationships between refugees and host countries. babu and blom (2014) introduce a model that seeks to delineate the key capacity components of a resilient food system, considering a country's capacity to create, manage, and utilise human resources for a resilient food system. significant challenges to aid provision include (a) harnessing the necessary stream of funding when grandstanding promises by countries are often never met; (b) establishing and coordinating the basic support network; (c) ensuring the logistics arrangements are effective, including communication networks; (d) prevention of resource misappropriation; (e) protection for officials and support workers on the ground; (f) protecting the vulnerable people needing aid; (g) operating with transparency and integrity; and (h) laying the structural and procedural foundations for self-reliance. effective lines of communication with donors and international agencies and charities are pivotal so that emergency arrangements can be established without delay and hindrance. these bodies need to deal with those individuals in the recipient countries truly knowledgeable about the capacity and specific problems facing food and water security, and fast-moving internal developments. the experts in the recipient countries must have the authority to be able to (a) quantify the levels of demand, (b) direct supplies, (c) recommend the siting of depots and distribution centres, and (d) highlight points of accessibility and vulnerability. in poorly governed countries, experts must be prepared to deal directly with these donors, agencies, and charities, difficult as that might be. the complexities of globalisation extend beyond food and water security (lerche 1998) . when people are deliberately persecuted, and honest law enforcement collapses, then non-partisan protection must be afforded, usually with outside security forces. unfortunately, ideological differences mean that the international community has often been shown to be ineffective in bringing about rapid termination of conflicts by imposing observers or armed forces, although thanks to relatively few major international donors, humanitarian relief has been forthcoming, albeit frequently late and inadequately funded. dealing with refugees and displaced people requires expertise and sympathetic support. housing provision together with monitoring and combatting infectious diseases, usually run in parallel with the issuance of food supplies. governments that prepare for worst-case scenarios are to be commended. even the distribution of authoritative guidance for populations in stress would represent a small step in the right direction, as would reinforcing the institutions that bind civil society, such as voluntary rescue and care organisations. networks of low-temperature clean and secure depots with associated distribution centres should be set up at the outset of disasters and conflicts. even in peaceful times, a marked cut in food waste helps food security. according to m. m. rutten (rutten 2013) around a third of the food for humans produced annually (about 1.3 billion tonnes) is either lost or wasted, and in developing economies the situation tends to be worse, with in excess of 40% lost during harvesting, processing and storage (fao. 2011. global food losses and food waste -extent, causes and prevention (see www.go. nature.com/um7vga). basic needs of refugees, as recommended by the unhcr and related organisations, are modest but are directly applicable to those displaced or besieged in their own country. unhcr recommends each refugee receive more than 2100 calories per day, recognising that a lack of food variety and inadequate supply of fruit and vegetables lead to deficiencies in essential vitamins and minerals. calorific intake can be reduced if the provided foodstuffs do not conform to traditional diets, or if the rations are traded to acquire other non-food goods and services. encouragement is needed to set up temporary gardens. fresh-water provision is of primary importance, with a minimum of 20 litres per person. a greater volume is needed, though, to prevent public-health problems of diarrhoea, cholera, and even polio. thus, clean-water sources and pumps are required along with taps within walking distance. vessels are needed for transfer and storage of water. water-purification tablets should be provided. sanitation systems are essential for hand washing and the safe disposal of urine, faeces, sanitary towels, wound dressings, infected and contaminated materials, and for the disposal of dead bodies. monitoring of faecal contamination is recommended. housing refugees and displaced people at short notice demands special expertise to avoid overcrowding and give adequate protection against inclement conditions. overlaying the fundamental needs for food, water, and shelter are meeting basic medical needs, particularly of the young, women, and the old and frail. in addition, within a short time, children require to be educated. host communities and host countries sometimes resist integration of forcibly deracinated people and can grow resentful at the costs incurred, especially if the host economy is weak. most financial assistance from donor countries is given to aid agencies rather than host countries. large-scale influxes of refugees can soon overwhelm the host country's infrastructural resources (chiefly fresh water, energy, housing, hospitals and healthcare systems, education, and waste disposal). other problems arise from combatants embedded in refugee cohorts, spreading the conflict and increasing policing costs. cultural incompatibilities between refugees and the host population create hostilities. refugees can suffer the dire consequences of being rendered stateless. in general, it is fair to say that humanitarian care is not able to sustain basic needs in the medium to long term. as a consequence of a funding crisis for humanitarian aid in the arab middle east, the world food programme was forced to suspend its desperately needed food-aid-voucher scheme for more than 1.6 million syrian refugees at the beginning of december 2014, the onset of winter. this suspension meant that refugees were less welcome in host countries and border closures are already being implemented in the immediate area as well as in the european union. axiomatically, just as responsible governments must be alert to and prepared for civil and other forms of unrest, they should always promote food production and remove any impediments to the uptake of improved technologies so that their economies have inbuilt resilience to dreadful events. likewise, governments should have in their ranks, or instantly available for consultation, competent scientists, technologists, and engineers able to advise on food, water, and energy resource distribution and allocation. over the past few decades, public-sector agricultural research and development in virtually all countries have suffered financial reductions and financial resources have been switched to activities regarded as more exciting and with greater wealth-creating potential; history shows this to be monumentally misguided. the urban disregard for agriculture is likely to continue as urbanisation increases, until the point food security threatens social stability. active or benign neglect of food-producing, food-processing, and food-distribution industries as well as of the scientists, technologists, and engineers underpinning its productivity, improvement and efficiency reveal incompetent governance. as an aside, the dearth of scientists, technologists, and engineers in active politics accounts for numerous policy failures. graduates in the arts (such as history and politics) and social sciences dominate politics and the upper echelons of the machinery of government (civil service) worldwide, people with little understanding or appreciation of business let alone of the "hard" sciences and engineering and their essential utility (and limitations) for mankind. perhaps this explains the growing dissatisfaction with the prevailing political classes. the scientific approach is that of the quest for knowledge by constantly questioning, developing and testing hypotheses by experimentation so that opinions change as "facts" change, oftentimes undermining policies that are not evidence-based, whereas many political parties are founded on inflexible belief systems, as are almost all religions. one aspect of food security in times of conflict and community disharmony has been the remarkable resilience of researchers to continue their studies or just maintain libraries, databases, records, laboratories, and genetic resources under the most trying conditions. the pursuit of knowledge is a fundamental feature of humans, as is the search for improvement. when there is blatant disregard of national constitutions as well as united nations treaties, protocols and conventions, and universities, colleges, schools, and research institutes become targets of malevolent forces, then the rest of the world must have no other option than to intervene, regardless of diplomatic niceties, in order to restore at least the vestiges of societal normality. as a first step, food security and the provision of fresh water for the besieged people must be a priority. if and when particularly large, heavily populated countries become embroiled in conflicts and/or major natural disasters, the existing international support efforts are likely to fail. this, in turn, may lead to a series of related conflicts, as opposing ideological pressures culminate in outright wars, invasions, and suffering on a huge scale. throughout the world, history has shown that unless they are relatively rich (and that may not be enough), smaller or militarily weak larger countries are influenced, for good or ill, by their more powerful and sometimes aggressive neighbours. as recent events demonstrate, conflicts in smaller countries rarely bring about rapid corrective measures from the international community, and adverse and damaging propaganda actively promoted in donor countries can prolong the suffering. ultimately though, food and fresh-water security are a prerequisite and eventually underpin stability, peaceful and thriving economies. today, much of the arab world is poorly governed and insecure for its citizens; they urgently deserve a better life. many of the most talented arabs seek a better life elsewhere. the warfare must be ended forthwith. grossly and unfairly misunderstood by much of the rest of the world, arabs demonstrate admirable resilience and stoicism yet retain their sense of humour tempered by understandable cynicism and justifiable suspicion of conspiracies. enemies of the arabs subject them to a tirade of insults and demeaning innuendos, often designed to deny them basic rights and international support. nonetheless, arabs must not be the continuing authors of their own misfortune, and a first step would be an end to internal conflicts followed by an effective region-wide clampdown on corruption at all levels. remember -it is the victor who determines the writing and shape of history. if the level of insecurity in the region were to get worse, then not only the arabs but also the rest of the world would pay a high price, so it is in everybody's interest to help restore peace. bluntly, the solution to their problems lies in the actions of the arabs themselves. in fully grasping the opportunities available through top-quality education , high standards of integrity and tolerance can be demanded from those in leadership roles in communities, organisations, businesses, and local and national government. ignorance can be reduced, even if not eliminated. essential components of democracy can be established, including independent and diverse news media, an autonomous judiciary operating to high standards of justice and unaffected by pressure groups and politicians, freedom of speech, and dynamic humanities and artistic sectors. wealth, and security of food, water, and energy, can and must be assured through the knowledge economy. harmony can be restored to communities suffering deep-seated divisions. furthermore, countries in the arab middle east will then be in a position to interact much more effectively and comprehensively in the international arena so that, if needed, external support and assistance can be fully and timeously harnessed. despite all the odds, this transition must be accelerated from the current dangerous condition to a much more enlightened and prosperous existence. education throughout society has proved to be a slow process, and can be resisted by regressive forces and indolence, so responsible leadership is a prerequisite. arab scientists, technologists, and engineers must contribute actively to this transition, thereby securing a safe, healthy, and buoyant future for all arabs. research and development priorities must be reassessed in the light of worsening nexus of water, food, and energy insecurity, and the desperate need to return to peaceful conditions. in legal jargon: time is of the essence. finally, a buoyant growth potential for the arab middle east is dependent on the fundamentals of demography, education, access to capital, technology, careful custodianship of its natural resources and environment, and social stability; all are threatened by this insecurity nexus. building capacity for resilient food systems human population reduction is not a quick fix for environmental problems the future of universities in the arab world. arab academy of science creating and evaluating accurate crispr-cas9 scalpels for genomic surgery food security policies for building resilience to conflict multiplex genome engineering using cripr/cas systems mapping of climate change threats and human development impacts in the arab region (51 pp.) food-and-nutrition-security food and agricultural organisation of the united nations action to build resilient livelihoods. prepared by the food and agricultural organization of the united nations (fao) for the world conference on disaster risk reduction world stabilization population unlikely this century global hunger index: the challenge of hidden hunger (56 pp.) value of traditional foods in meeting macro-and micronutrient needs: the wild plant connection overview of the roles of energy and water in addressing global food security an overview of mitigation and adaptation processes and strategies to address the impacts of climate change on food, water, and energy security in the arab middle east a programmable dual-rna-guided dna endonucleases in adaptive bacterial immunity a meta-analysis of the impacts of genetically modified crops the conflicts of globalization resilience for food security in refugee-hosting communities international budget partnership. www.internationalbudget.org organisation of economic co-operation and development economics of salt-induced land degradation and restoration what economic theory tells us about the impacts of reducing food losses and/or waste: implications for research, policy and practice. agriculture and food security national association of state universities and land-grant colleges, experiment station committee on organization and policy, a science roadmap for the future. www.nasulgc.org/comm_food.htm the economist commodity-price index a gap analysis methodologhy for collecting crop genepools: a case study with phaseolus beans key: cord-345591-zwh1xj5u authors: al-dorzi, hasan m.; aldawood, abdulaziz s.; khan, raymond; baharoon, salim; alchin, john d.; matroud, amal a.; al johany, sameera m.; balkhy, hanan h.; arabi, yaseen m. title: the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study date: 2016-10-24 journal: ann intensive care doi: 10.1186/s13613-016-0203-z sha: doc_id: 345591 cord_uid: zwh1xj5u background: middle east respiratory syndrome coronavirus (mers-cov) has caused several hospital outbreaks, including a major outbreak at king abdulaziz medical city, a 940-bed tertiary-care hospital in riyadh, saudi arabia (august–september 2015). to learn from our experience, we described the critical care response to the outbreak. methods: this observational study was conducted at the intensive care department which covered 5 icus with 60 single-bedded rooms. we described qualitatively and, as applicable, quantitatively the response of intensive care services to the outbreak. the clinical course and outcomes of healthcare workers (hcws) who had mers were noted. results: sixty-three mers patients were admitted to 3 mers-designated icus during the outbreak (peak census = 27 patients on august 25, 2015, and the last new case on september 13, 2015). most patients had multiorgan failure. eight hcws had mers requiring icu admission (median stay = 28 days): seven developed acute respiratory distress syndrome, four were treated with prone positioning, four needed continuous renal replacement therapy and one had extracorporeal membrane oxygenation. the hospital mortality of icu mers patients was 63.4 % (0 % for the hcws). in response to the outbreak, the number of negative-pressure rooms was increased from 14 to 38 rooms in 3 mers-designated icus. patients were managed with a nurse-to-patient ratio of 1:0.8. infection prevention practices were intensified. as a surrogate, surface disinfectant and hand hygiene gel consumption increased by ~30 % and 17 n95 masks were used per patient/day on average. family visits were restricted to 2 h/day. although most icu staff expressed concerns about acquiring mers, all reported to work normally. during the outbreak, 27.0 % of nurses and 18.4 % of physicians working in the mers-designated icus reported upper respiratory symptoms, and were tested for mers-cov. only 2/196 (1.0 %) icu nurses and 1/80 (1.3 %) physician tested positive, had mild disease and recovered fully. the total sick leave duration was 138 days for nurses and 30 days for physicians. conclusions: our hospital outbreak of mers resulted in 63 patients requiring organ support and prolonged icu stay with a high mortality rate. the icu response required careful facility and staff management and proper infection control and prevention practices. the middle east respiratory syndrome (mers) coronavirus is a recently identified virus that is closely related to the severe acute respiratory syndrome coronavirus (sars-cov) [1] , causes severe hypoxemic respiratory failure with multiorgan failure and frequently requires admission to the intensive care unit (icu) [2, 3] . as of september 27, 2016 , the world health organization (who) reported 1806 laboratory-confirmed cases, including 643 related deaths [4] . the majority (~80 %) of mers cases occurred in saudi arabia [4] , where several hospital outbreaks happened [5, 6] with 45 % of all cases taking place within healthcare facilities [7] . the outbreak in the republic of korea illustrated the global threat of this disease. it started in may 2015 and resulted from a case with travel history to the arabian peninsula [4] . human-to-human transmission occurred to close contacts [family members, other patients and healthcare workers (hcws)] and led to 186 cases of mers-cov infections with 19 % fatality rate. in our hospital, king abdulaziz medical city-riyadh, an outbreak of mers disease occurred in august to september 2015 [4] , led to significant disruption of hospital functions and resulted in 130 mers cases [8] with 63 patients requiring icu admission. the outbreak was attributed to crowding, movement of infected but undiagnosed patients and breaches in infection prevention and control practices [4] . details of the hospital outbreak have been published elsewhere [8] . most of the medical literature on mers has focused on describing the characteristics and outcomes of affected patients. preparedness and the response of the healthcare system at its different levels are crucial to contain this disease and manage its associated outbreaks. nevertheless, little is published about how the healthcare system responded to the disease and hospital outbreaks. the objective of this study was to describe the response of the icu to a hospital mers outbreak, the associated changes in its workflow and the impact on its hcws. this study was an observational study conducted at the intensive care department of king abdulaziz medical city, a 940-bed tertiary-care referral hospital in riyadh, saudi arabia, that was accredited by the joint commission international. the hospital had an infection prevention and control department. the intensive care department covered 5 units: an 8-bed trauma icu (unit a), a 21-bed medical-surgical intensive care unit (unit b), a 9-bed surgical icu (unit c), an 8-bed neurologic icu (unit d) and a 14-bed stepdown unit (unit e). the department also provided coverage to boarding patients in the 15-bed resuscitation area in the emergency department (ed). the hospital had also an 8-bed burn icu. the icus were operated as closed units with 24-h, 7-day onsite coverage by boardcertified critical care intensivists [9] . normally, 5 medical teams covered the units during the day with each team consisting of one intensivist consultant, one registrar/ fellow and 1-3 residents. the nurse-to-patient ratio in all the icus was mostly 1:1. one certified respiratory therapist covered a maximum of six ventilated patients. additionally, the department had a rapid response team, which covered the hospital wards and was activated according to predefined criteria and is covered by a sixth separate team [10] . the department has had several ongoing quality improvement projects and indicators. infection prevention and control practices, such as hand hygiene and the ventilator care bundle, were monitored by multidisciplinary icu teams and the infection prevention and control department [11] . for intubated patients, ventilator care was provided by specialized respiratory therapists and included using closed endotracheal suctioning systems which were changed every 72 h or as clinically indicated [12] , changing the ventilator circuits in between patients or if they became soiled or damaged [12] , and using heat and moisture exchangers which were changed every 7 days or when visibly soiled [12] . due to the high prevalence of multidrug-resistant organisms and prior cases of influenza and mers infection in the hospital, droplet precautions were applied to all icu patients since february 2012 [11] . sporadic cases of mers cases have been managed in our icu since february 2013. the characteristics, management and outcomes of the initial mers cases managed in our unit were described previously [3] . all hcws were required to undergo fit testing for the n95 respirators (table 1 ). an infectious disease epidemic plan (idep) was initially released in may 2014 and was revised in march 2015. table 1 describes selected plan elements. according to the idep, one unit (unit a) was designated as the primary receiving unit for mers patients, because of its geographic location being away from main hospital traffic and because 7 of its 8 rooms were negative-pressure airborne infection isolation rooms (aiir). the plan was not explicit about which units would be used if the number of cases exceeded the capacity of this unit. however, unit b had 4 negative-pressure rooms. in our hospital, mers was suspected based on clinical presentation and confirmed by laboratory testing as recommended by the who and the saudi ministry of health [4, 13] . in our icu, the workup of patients having lower respiratory tract infections was standardized to include bacterial gram stain and culture, mers-cov polymerase chain reaction (pcr), h1n1 pcr, bacterial and viral multiplex pcr on respiratory samples, mycoplasma, chlamydia and legionella serology and, if suspected, tuberculosis workup. the respiratory samples were routinely obtained by blind deep tracheal aspirate in intubated patients. in the hospital biosafety level 2 laboratory, mers-cov screening was performed by real-time reverse-transcription (rrt) pcr on respiratory samples by checking for the upstream e protein genome (roche modular dx coronavirus) and infection confirmation by detecting the open reading frame 1a genome (mers-cov kit from tib molbiol) [14] . laboratory workers were fit-tested and applied n95 respirators while handing respiratory samples. positive samples were sent to the saudi ministry of health reference laboratory for confirmation. viral culture was not performed as biosafety level 3 is needed. we noted mers cases admitted to the icu from july 1 to october 21, 2015, and collected data on the clinical characteristics, management and outcomes of the affected hcws. we also obtained data about the rrt-pcr performed for icu patients. we identified the physicians and nurses who reported sick leave for respiratory illnesses. as surrogate for infection control practices, we obtained data on the related consumables for units a and b before and during the outbreak (aprilseptember 30, 2015) . we also collected qualitatively our own observations and those of other hcws on the icu response during the outbreak using interviews and open discussions. descriptive data were presented as means and standard deviations or frequencies and percentages, as appropriate. the infection prevention and control consumables were compared in the 4 months before (april to july) and the 2 months during (august and september) the outbreak. the plan will be activated by the chair of the outbreak response committee based on the phase definition phase i 0-5 cases of suspected or confirmed in the hospital phase ii 6-30 cases of suspected or confirmed in the hospital phase iii >30 cases of suspected or confirmed in the hospital phase i confirmed mers-cov cases requiring intubation will be assigned a negative-pressure room and cohorted in one icu confirmed cases that have been diagnosed with mers-cov in any icu other than the trauma icu (unit a), shall be transferred to the trauma icu (unit a) as soon as possible. phase ii all mers-cov patients will be cohorted in one unit. if the number of patients exceeds its capacity, then other units are identified to receive the additional cases closure of all nonessential hospital functions phase i all services run without interruptions except for certain precautions for mers patients phase ii all elective surgery shall be canceled to free more icu beds phase iii all elective cardiac surgery shall be canceled outpatient clinic visits shall be limited to urgent visits only healthcare worker (hcw) management all hcws shall be aware of 1. relevant infection prevention and control policies and procedures 2. their annual influenza immunization status. if not vaccinated, please contact the employee health clinic to arrange for an appointment 3. their n95 fit check/test status. if have not been fit-tested, please contact the employee health clinic to arrange for an appointment hcws exposed to a confirmed mers-cov case shall be assessed according to a predetermined protocol hcws requiring isolation at home and happen to share a room with another hcw will be provided a room in the designated accommodation for isolation till cleared by the infection prevention and control department an infection prevention and control officer is available 24 h per day, 7 days per week in july 2015, five cases of acute respiratory failure were referred to the icu team from the ed and wards and were diagnosed to have mers pneumonia. as the number of mers patients increased, the idep was activated on august 2, and included strict implementation of infection control measures, including airborne and contact isolation for confirmed and probable mers cases, and droplet and contact isolation for suspected cases [8] . on august 18, phase iii of the idep was activated (table 1) , which included ed closure, elective surgical procedure cancelation and outpatient clinic suspension [8] . meanwhile, the icu maintained full operations. figure 1 suspected mers cases had rrt-pcr on nasopharyngeal swabs in nonintubated patients and on deep tracheal aspirates in intubated patients. fiberoptic bronchoscopy was not performed for the diagnostic workup of mers or ventilator-associated pneumonia. repeated testing was frequently needed to make the diagnosis. among our 63 critically ill mers patients, the initial mers-cov testing was performed on nasopharyngeal swabs in 29 and deep tracheal aspirates in the rest (n = 34). in the first sample, mers-cov was detected in only 6/29 (20.7 %) nasopharyngeal samples and 26/34 (76.5 %) deep tracheal aspirates. after initial negative or equivocal nasopharyngeal swabs (n = 23), a second nasopharyngeal swab was performed in 16 patients (positive in 37.5 %) and deep tracheal aspirate in 5 (positive in 100 %). our microbiology laboratory extended its working hours and prioritized testing samples coming from the icu and the rest of the hospital. the number of mers-cov tests performed on icu patients went up from an average of 1.5 before to 5.3 per day during the outbreak with a maximum of 19 tests on september 4, 2015. in patients with suspected mers and negative rrt-pcr, the test was repeated after 1-2 days. there was a general consensus among our intensivists that three negative lower respiratory samples and low clinical pretest probability were needed to exclude mers-cov infection. for patients with confirmed mers, the test was repeated twice weekly until 3 consecutive tests were negative. the mean age of the 63 patients was 57.9 ± 18.6 years with the majority (69.8 %) being males. eight hospital workers required icu admission after acquiring mers. table 2 summarizes their characteristics. half of them did not have direct contact with patients. one of them was a pregnant nurse that worked in the ed. all but one required intubation. the medical management of mers patients was largely supportive. most (80.9 %) mers patients required endotracheal intubation, which was performed by the most experienced available physician with airborne precautions. lung-protective ventilation was implemented for acute respiratory distress syndrome with tidal volumes (4-6 ml/kg of ideal body weight). to reduce the airborne generating procedures, we discouraged the use of noninvasive ventilation for suspected mers cases. nevertheless, it was used in the initial management of 12 (19.0 %) patients. these patients were either suspected to have concomitant cardiogenic pulmonary edema or had milder disease. intubation was needed for 10 patients. highflow oxygen therapy was not used as it was unavailable. when needed, bronchodilators were used via metered dose inhalers rather than nebulizers. most (58.7 %) mers patients required vasopressors. renal replacement therapy was provided for 37 (58.7 %) patients. for the hospital workers acquiring mers (n = 8), cisatracurium infusion was used in 7 (87.5 %), early prone positioning in 4 (50 %), continuous renal replacement therapy in 4 (50 %) and extracorporeal membrane oxygenation in 1 ( table 2) . none of the patients received ribavirin, interferon therapy or high-dose steroids. the hospital mortality of mers patients was 63.4 % with all deaths occurring in the icu. all hospital workers who had mers survived and were discharged to home. the icu and hospital length of stay were prolonged (15.0 ± 17.0 and 30.6 ± 23.1 days, respectively). during the outbreak, our hospital established a command center, which met twice daily, and oversaw all interventions in accordance to the idep. the intensive care department chairman was a member of the command center and presented daily the number of suspected and confirmed cases in the icu, bed and staff management issues and any challenges. the department chairman attended all morning handover meetings in the icu where he received input from the icu teams and provided feedback from the command center. the hospital provided an intranet page that had educational material on mers, mers management guidelines and proper infection control practices. the page was regularly updated. additionally, the hospital frequently informed staff about the mers outbreak status through emails. staff could communicate with the command center regarding any outbreak-related concern or question. the intensive care department communicated with the medical staff about the saudi ministry of health and who practice guidelines. before and during the outbreak, the who interim guidance for the management of suspected and confirmed mers-cov infection [15] was circulated to our icu staff. moreover, a letter expressing gratitude and encouragement was sent from the department chairman to all hcws. family visiting to mers patients was restricted from an open visiting policy to 2 h per day during the evening with visitors not allowed to enter patient rooms. visiting outside these hours was allowed in selected cases if the clinical condition required. to update the patients' families, the icu consultant contacted and updated the next of kin by phone every day and addressed the family concerns. a nurse was assigned to screen all staff and visitors entering each unit by asking for symptoms of acute respiratory infection and measuring temperature. staff and family members with symptoms of acute respiratory illness or fever were not allowed to enter. the initial mers cases were admitted to the designated mers unit (unit a). as the number of patients increased, the icu leadership identified other icus as potential placement units. the hospital clinical engineers converted a total of 24 standard rooms in unit b and unit c to negative-pressure rooms by increasing air exhaust more than supply by 50 cubic feet per minute. as the number of suspected and confirmed mers patients increased, unit b and then unit c were used. as the number of our mers patients increased beyond unit a capacity, patients without mers were transferred to other units or hospitals to increase bed capacity. the old pediatric icu, which was recently vacated in june 2015 after the opening of a new pediatric hospital, was used to care for stable and long-term patients. during the outbreak, 13 and 7 patients from units a and b, respectively, were transferred to the other icus (units c-e and the old pediatric icu), and 1 patient to another hospital. the care for mers patients was demanding. for example, 4 of the 8 affected hcws required prone positioning for the management of acute respiratory distress syndrome ( table 2 ). also 4 of them required continuous table 2) . the care was also associated with significant exposure risk. this can be reflected in the duration of mechanical ventilation for the hcws who required intubation (7-57 days) and length of icu stay (7-63 days) ( table 2) . during the outbreak, the nurse-to-patient ratio was mostly 1:1 except for one patient on ecmo (2:1). additionally, 1-2 additional nurses were deployed in each unit to assist in procedures such as prone positioning and to monitor and correct infection control practices. in unit a, for instance, the nurse-to-patient ratio was 1:1.2 before the outbreak and became 1:0.8 during the outbreak. we restricted medical management to the attending physicians, senior registrars and critical care fellows. rotating residents were not allowed to work in the icu during the outbreak. entry of nonclinical staff, such as research coordinators, was restricted and the ongoing clinical trials were put on hold, except for mers studies, to reduce staff exposure. during the outbreak, concerns among icu staff were raised about acquiring mers and transmitting the virus to their families; a concern that was substantiated by seeing hospital workers infected and developing critical illness. however, many felt privileged to be part icu team managing the outbreak and taking care of mers patients; none refused to report to work as per schedule. two pregnant icu nurses were redeployed to low-risk units. staff members who developed fever, respiratory symptoms or gastrointestinal illness were asked not to present to work, but rather to report to the ed or the employee health clinic depending on their illness severity. of the 152 bedside nurses covering units a and b, 41 (27.0 %) nurses had symptoms of acute respiratory infection during the outbreak and consequently had nasopharyngeal swabs obtained for mers-cov; all tested negative. their total sick leave duration was 138 days (range: 1-15 days per nurse). in comparison, in the 2 months before the outbreak, 26 (17.1 %) nurses had sick leaves for a total of 33 days (range 1-2 days per nurse). of the 49 nonresident icu physicians, 9 (18.4 %) physicians received sick leave for a total of 30 days (range 3-4 days per physician). in unit c, two nurses (1.0 %) of 196 nurses who worked in mers units (units a, b and c) and one rotating resident (1.3 %) out of 80 physicians covering the icus tested positive for mers-cov. the resident and one of the nurses were symptomatic and required hospitalization in a mers ward for approximately 1 week and both recovered fully. in february 2012, long before the mers outbreak, droplet precautions had been added to the standard precautions for all icu patients, mainly to prevent the transmission of influenza. during the outbreak, airborne precautions were added for all confirmed and suspected mers cases. although all staff were required to be fit-tested for the n95 respirators before the outbreak (table 1) , we discovered that many icu staff were not tested. during the outbreak, a clinic was emergently opened to fit test hcws and the results were documented. specific policies and procedures were developed or updated for donning and doffing personal protective equipment (ppe). related visual instructions were provided inside each icu room. outside patient rooms, carts containing ppe were organized to facilitate donning in the correct sequence. during the outbreak, additional training on hand hygiene techniques and ppe application was provided. housekeepers were also retrained on proper cleaning techniques and ppe use. the intensive care department worked closely with the infection prevention and control department on all aspects of infection control. the implementation of such infection control measures required having adequate ppe supplies, such as respirators, goggles, face shields and gowns. table 3 describes the consumption of surface disinfectants, antiseptic alcohol for hand hygiene, n95 masks and other ppe before and during the mers-cov outbreak. during the outbreak, the consumption of detergent surface disinfectant and ethyl alcohol for alcohol-based hand rub increased by almost 30 % and the use of n95 masks increased by >15 times compared to the preceding 4 months. the number of examination gowns per patient per day decreased during the outbreak probably due to staff avoiding unnecessary exposure. twenty-four powered air-purifying respirators were made available to staff who failed the n95 respirator fit test. they were used by 8 physicians, 7 nurses and 14 respiratory therapists. training sessions on their application were conducted. in this report, we described how the icu responded to a mers-cov outbreak at a tertiary-care hospital. the outbreak led to 63 mers patients requiring prolonged icu care and most received invasive mechanical ventilation, vasopressors and renal replacement therapy. the overall mortality was 63 %, but all affected hospital workers survived. the outbreak management included almost tripling the icu capacity of negative-pressure rooms and intensifying infection prevention and control practices. even though icu staff had significant exposure risk, very small number acquired mers-cov. response to incidents such as an infectious hospital outbreak requires a robust hospital-wide command and control structure that is able to make rapid informed decisions across an institution. as it was the case in our hospital, the control of outbreak may require major interventions such as closing the ed, suspending elective surgeries, preventing inter-facility patient transfers, canceling ambulatory clinics and outpatient diagnostic procedures, preventing hospital staff from working at other institutions and restricting hospital visitors [16] . our hospital had a preexisting idep, which facilitated managing and containing the mers-cov outbreak. such a plan is mandatory for every hospital. mers infection is associated with several challenges. its presenting symptoms overlap with those of other severe acute respiratory illnesses and include fever (71 %), cough (68 %), dyspnea (66 %) and diarrhea (32 %) [17] , often in older adults with preexisting chronic comorbidities [17, 18] . common laboratory abnormalities include leukopenia, lymphocytopenia, thrombocytopenia and elevated serum creatinine, lactate dehydrogenase, and liver enzymes [17] . the initial chest radiographs show minimal abnormality to extensive bilateral infiltrates [17] . unfortunately, many frontline physicians are unfamiliar with the mers case definition, probably because cases are sporadic, leading to delayed or even missed diagnosis. delayed recognition may lead to exposing many other patients, visitors and hcws to the infection as it was the case in our hospital. mers-cov nosocomial transmission is thought to be via respiratory droplets, and contact spread is suspected [19] . in korea, the delayed diagnosis of an infected traveler to the arabian peninsula led to 186 mers cases and resulted in intraand inter-hospital transmission [20] . in saudi arabia, 45 % of mers cases were acquired in the healthcare setting with 12 % of all cases being hcws [7] . therefore, taking a detailed history, knowing mers case definitions, standardizing pneumonia workup, obtaining lower respiratory tract specimens [21] and implementing droplet isolation for suspected cases are crucial interventions to break the transmission chain in the healthcare setting. admitting mers patients in single-bedded negativepressure rooms and cohorting them in selected units are recommended to facilitate providing care and monitoring [22] . during an outbreak, clinical engineering should have expedient plans to convert standard rooms. retrofitting the rooms with externally exhausted hepa filters may be a solution [16] . outbreaks can lead to significant increase in the need for icu beds, but may simultaneously reduce the available beds. the sars outbreak in toronto led to 10-day closures of 35 icu beds, which represented 38 % of the tertiary-care university medical-surgical icu beds in toronto [16] . hence, hospitals should always have plans to augment icu bed capacity, such as by transforming general wards. the ability of any hospital to deal with an infectious outbreak is decided by the availability of icu beds [23] . caring for mers patients represents a substantial exposure risk for icu staff because of three reasons: high exposure dose, long daily contact hours and prolonged icu stay with viral shedding. mers-cov patients requiring icu admission have higher viral load than other mers patients [24] . aerosol-generating procedures, such as noninvasive ventilation, suctioning and bronchoscopy, add to the exposure and transmission risk [25] . extended bedside care is needed for mers patients due to the requirement of organ support such as mechanical ventilation, vasopressor therapy, continuous renal replacement therapy, prone positioning and extracorporeal membrane oxygenation [2, 3] . in this study, we observed an increase in the nurse-to-patient ratio from 1:1.2 to approximately 1:0.8 during the outbreak. the sars epidemic was also associated with increases in the nurse-topatient ratio [26] . stay in the icu can last for weeks [3] , which we observed. additionally, mers-cov shedding can be prolonged and may last for >30 days [27] . the exposure risk to mers-cov can exert significant psychosocial stress on hcws. death has occurred in young hcws who acquired mers-cov infection [28] , which adds to this fear. during the sars outbreak in toronto, a survey of hcws found that >60 % of respondents reported sars-related concern for their own or their family's health [29] . moreover, 29 % of respondents had probable emotional distress [29] . during our outbreak, many icu staff expressed concerns about acquiring mers. staff safety should be a primary goal in a hospital infectious outbreak. pregnant and immunocompromised staff should be redeployed to lower-risk areas [30] , which we did. proper exposure management should be pre-planned, which was determined in our idep. n95 respirator fit testing should be performed at hiring of new staff and done for all other staff before any outbreak. although fit testing was required for all staff, many did not have fit testing done. however, in response to the outbreak, fit testing was performed to all staff and powered air-purifying respirators were provided to those who failed fit testing. strict infection prevention and control practices should be implemented and audited. this was performed in our units. repetitive training is recommended [31] . despite intensive infection prevention practices, 3 of our icu staff had mers-cov infection during the outbreak likely due to suboptimal ppe use while intubating yet undiagnosed patients. mers management was supportive and largely adhered to the who recommendations [15] . it included intubation, early prone positioning and neuromuscular blockade for moderate-to-severe acute respiratory distress syndrome as these interventions have been shown to improve the outcomes of ards patients [32, 33] . although noninvasive ventilation use was discouraged, it was used in 12 patients. the who considers noninvasive ventilation an option in selected mers cases [15] . it should be used as a short trial without delaying intubation if unsuccessful [15] . moreover, high-flow oxygen by nasal cannula may be another option [15, 34] ; however, the associated transmission risk as a result of aerosol generation is unknown. systematic corticosteroids, ribavirin and interferon were avoided as they have no proven benefit [15, 35] . our mers patients had high mortality (63 %). the previously reported mortality of mers patients who had critical illness ranged from 58 to 84 % [2, 3, 18] . none of our hcws who developed mers died, which was gratifying to our staff. our mers-cov hospital outbreak stressed our system to unprecedented limits. we learned many lessons from it ( table 4 ). the successful management of outbreak required integrating icu functions with the hospitalwide plans, having preparedness plans, implementing proper infection control practices and managing staffing and staff exposure. every hospital should have an infectious disease epidemic plan that should govern the response to an infectious disease outbreak. the response should cover organizing patient services, implementing infection control, managing employee exposure and communicating with national health services and with hospital staff hospital leaders should be prepared to increase the capacity of negative-pressure airborne infection isolation rooms in the case of an infectious disease outbreak all healthcare workers should receive training on proper hand hygiene and personal protective equipment application. hand hygiene and personal protective equipment practices should be monitored. education should be repeated periodically all healthcare workers should be fit-tested for n95 respirators on hire with the result documented in their files. periodic audit of this requirement should be done hospitals should make plans to acutely increase personal protective equipment supplies as consumption increases tremendously during an infectious disease outbreak hospital and icu leaders should have plans to cover healthcare workers who are exposed or become symptomatic to avoid potential staff shortage severe acute respiratory syndrome vs. the middle east respiratory syndrome clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities the national command control center; ministry of health-kingdom of saudi arabia. mers-cov in ksa notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia weekend and weeknight admissions have the same outcome of weekday admissions to an intensive care unit with onsite intensivist coverage impact of an intensivist-led multidisciplinary extended rapid response team on hospital-wide cardiopulmonary arrests and mortality a multifaceted approach to improve hand hygiene practices in the adult intensive care unit of a tertiary-care center comprehensive evidence-based clinical practice guidelines for ventilator-associated pneumonia: diagnosis and treatment ministry of health kingdom of saudi arabia. infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection assays for laboratory confirmation of novel human coronavirus (hcovemc) infections clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected-interim guidance identification and containment of an outbreak of sars in a community hospital middle east respiratory syndrome: knowledge to date middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions mers outbreak in korea: hospital-to-hospital transmission an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus hospital preparedness and sars association of higher mers-cov virus load with severe disease and death, saudi arabia aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review clinical review: sars-lessons in disaster management middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications middle east respiratory syndrome coronavirus infections in health care workers psychosocial effects of sars on hospital staff: survey of a large tertiary care institution sars and hospital priority setting: a qualitative case study and evaluation infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease prone positioning in severe acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study none. the authors declare that they have no competing interests. written informed consent was not obtained for publication of these data. the institutional review board of the ministry of national guard health affairs approved the retrospective clinical data collection on mers patients, and no consents were required. abbreviations ed: emergency department; hcw: healthcare worker; icu: intensive care unit; idep: infectious disease epidemic plan; mers: middle east respiratory syndrome; ppe: personal protective equipment; rrt-pcr: real-time reversetranscription polymerase chain reaction; who: world health organization. hmd was involved in conception and design, data collection, statistical analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content and approval of the final version to be published. asa contributed to the analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. rk helped in data collection, analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. sb contributed to the analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. jda helped in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. aam was involved in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. smj helped in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. hhb contributed to data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. yma helped in conception and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content and approval of the final version to be published. all authors read and approved the final manuscript.author details 1 icu2 and ticu, intensive care department, king abdulaziz medical city, king key: cord-329190-kv9n2qj3 authors: rabaan, ali a.; alahmed, shamsah h.; bazzi, ali m.; alhani, hatem m. title: a review of candidate therapies for middle east respiratory syndrome from a molecular perspective date: 2017-09-01 journal: journal of medical microbiology doi: 10.1099/jmm.0.000565 sha: doc_id: 329190 cord_uid: kv9n2qj3 there have been 2040 laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) in 27 countries, with a mortality rate of 34.9 %. there is no specific therapy. the current therapies have mainly been adapted from severe acute respiratory syndrome (sars-cov) treatments, including broad-spectrum antibiotics, corticosteroids, interferons, ribavirin, lopinavir–ritonavir or mycophenolate mofetil, and have not been subject to well-organized clinical trials. the development of specific therapies and vaccines is therefore urgently required. we examine existing and potential therapies and vaccines from a molecular perspective. these include viral s protein targeting; inhibitors of host proteases, including tmprss2, cathepsin l and furin protease, and of viral m(pro) and the pl(pro) proteases; convalescent plasma; and vaccine candidates. the medline database was searched using combinations and variations of terms, including ‘middle east respiratory syndrome coronavirus’, ‘mers-cov’, ‘sars’, ‘therapy’, ‘molecular’, ‘vaccine’, ‘prophylactic’, ‘s protein’, ‘dpp4’, ‘heptad repeat’, ‘protease’, ‘inhibitor’, ‘anti-viral’, ‘broad-spectrum’, ‘interferon’, ‘convalescent plasma’, ‘lopinavir ritonavir’, ‘antibodies’, ‘antiviral peptides’ and ‘live attenuated viruses’. there are many options for the development of mers-cov-specific therapies. currently, mers-cov is not considered to have pandemic potential. however, the high mortality rate and potential for mutations that could increase transmissibility give urgency to the search for direct, effective therapies. well-designed and controlled clinical trials are needed, both for existing therapies and for prospective direct therapies. middle east respiratory syndrome coronavirus overview middle east respiratory syndrome coronavirus (mers-cov) was first isolated in jeddah in the kingdom of saudi arabia (ksa) from a 60-year-old male hospital patient, who died 24 june 2012, 11 days after presenting with acute pneumonia and subsequent renal failure [1] . since then, the who have been notified of 2040 laboratory-confirmed cases, including 712 deaths [2] . while most cases have arisen in the middle east, cases have also emerged in 27 countries worldwide in travellers from the middle east and/ or in their contacts [2] . most human mers-cov infections are considered to be the result of multiple zoonotic transfers. bats are the most likely mers-cov natural reservoir, as with other mammalian coronaviruses (covs), while camels are likely to be the major zoonotic source for human infections [3] [4] [5] . secondary human-to-human transmission is considered to be limited, occurring mainly within family and healthcare settings. the first cluster of cases in humans was retrospectively identified to have occurred in a public hospital in jordan in april 2012 [6] . multiple healthcare facility-associated outbreaks have since occurred in the middle east, most notably in ksa, often linked to deficiencies in infection control procedures [7] [8] [9] [10] [11] [12] [13] . although cases outside the middle east have mainly been isolated, a large outbreak occurred in korea in june 2015, in which human-human transmission resulted in 186 cases and 36 deaths [14] . increased vulnerability to either cross-species or trans-human transmission could result from viral adaptations [15] . mers-cov infection is often accompanied by acute viral pneumonia, and sometimes gastrointestinal symptoms. clinical severity varies from asymptomatic to death, and the extent of asymptomatic spread is unclear. the high mortality rate is mainly accounted for by acute respiratory distress syndrome (ards) [7, 15, 16] . higher mortality is observed among vulnerable patients, such as older individuals and those suffering from comorbid illness, and is also associated with high viral load [7, 11, 15, 17] . in one study in a ksa hospital, intensive care unit (icu) admission among mers-cov patients was associated with a mortality rate of 74.2 % [11] . while mers-cov is not currently considered to have pandemic potential, it is clear that human-human transmission does occur. the exact mechanisms by which mers-cov is transmitted from animals to humans have not been fully elucidated. in the south korean outbreak, the virus emerged in second-and third-generation contacts, resulting in the first human case to be imported into china [18] . this raised concern that viral mutations were contributing to humanhuman transmission. given its high mortality and poor outcomes for vulnerable patients, and the potential for viral mutations, there is no room for complacency in the search for therapeutic options for mers-cov. there is currently no specific therapy. many of the therapeutic options used have been adapted from approaches used to treat severe acute respiratory syndrome (sars-cov) during the outbreak of 2003, and/or the h1n1 influenza virus during the outbreak of 2009 [19] . however, while mers-cov and sars-cov are phylogenetically related betacoronaviruses, they differ in many important respects. mers-cov utilizes human dipeptidyl peptidase 4 (dpp4; cd26) receptors, with binding mediated by the viral spike (s) protein, not the angiotensin-converting enzyme 2 (ace-2) receptors used by sars [20] [21] [22] [23] [24] . mers-cov also has a wider cellular tropism [24] [25] [26] . therapies currently used include broad-spectrum antibiotics, corticosteroids and anti-viral treatments, such as interferons (ifn), ribavirin, lopinavir-ritonavir, or mycophenolate mofetil [19, 22, [27] [28] [29] [30] [31] . however, the efficacy and/or safety of many of these therapies is unclear, and none are specific to mers-cov. ribavirin monotherapy, for example, is associated with multiple side-effects in the treatment of other viral illnesses, including sars-cov, has uncertain efficacy, and has not been tested in animal studies or randomized control trials for mers-cov [22, 31, 32] . corticosteroids have not been successful in the treatment of respiratory distress or lung fibrosis in mers-cov [31, 32] . meanwhile, studies in sars-cov and h1n1 patients suggest that corticosteroid use may in fact increase viral replication in airways, and sars patient and animal studies indicate that it contributes to immunosuppression [33] [34] [35] . mycophenolate mofetil has been associated with fatal disease and high viral loads in a marmoset model of mers-cov infection [36] . ifn therapy, alone or in combination with ribavirin or lopinavir-ritonavir, has shown greater promise in in vitro, animal and human studies [37] [38] [39] [40] . however, clinical studies on ifns vary with respect to factors such as time of administration and type of patient [19, 22, 40] . overall, there is a lack of randomized control trials (rcts) designed to test the safety and efficacy of any potential therapies specific to mers-cov, and much of the information available for existing therapies is based on in vitro and/or animal studies [22, 40, 41] . a position paper on the evidence base for specific mers-cov therapies, published by public health england (phe) and the world health organization-international severe acute respiratory and emerging infection consortium (isaric-who), suggested that benefit was likely to exceed risk for convalescent plasma, lopinavir-ritonavir, ifns and monoclonal/polyclonal antibodies, while, by contrast, for ribavirin monotherapy and corticosteroids it was considered that the risks would outweigh the benefits [42] . for interferon/ribavirin combination therapy, nitazoxanide and chloroquine, the available data were considered to be inadequate for assessment [42] . in this review, we consider potentially effective mers-cov therapies, including ifns, lopinavir-ritonavir and inhibitors of proteases, including tmprss2 and cathepsin l, as well as mers-cov-specific potential therapies, including convalescent plasma, monoclonal antibodies (mabs), antiviral peptides and candidate vaccines. these therapies will be considered from a molecular perspective, in the context of the infection and replication mechanisms of mers-cov. the therapies are summarized in table 1 . mers-cov lineage and structure mers-cov is a betacoronavirus belonging to clade c (lineage 3) of the betacoronaviruses [43] . its closest known coronavirus relatives are the prototypic clade c betacoronaviruses, tylonycteris bat virus hku4, pipistrellus bat hku5 virus and neoromicia zuluensis bat pml/2011 (neocov) virus [1, [43] [44] [45] [46] [47] . in common with other coronaviruses, the genome of mers-cov is a single, positive-stranded rna of over 30 000 nucleotides. it encodes 10 predicted open reading frames (orfs) and genes for 4 structural proteins, namely the spike (s), nucleocapsid (n), membrane (m) and envelope (e) proteins (figs 1 and 2) [48] [49] [50] . orf 1a and 1b encode virus replication-related proteins (pp1a, pp1ab), which are cleaved to give 16 non-structural proteins (nsps) involved in synthesis of viral rna and recombination ( fig. 2) [48] [49] [50] . these include nsp-14, which contains a 39-to-59 exoribonuclease (exon) domain that is important in viral proofreading and in determining the sensitivity of rna viruses to mutagens. thus small-molecule inhibitors references s1/dpp4 binding antibody (mouse): s1 rbd in vitro [76] antibody (human): s1 rbd m336, m337, m338 in vitro/in vivo (mouse, rabbit -m336) [77] [78] [79] antibody (human): s1 rbd in vitro [80, 81] antibody (mouse-humanized): s1 rbd in vitro/in vivo (mouse) prophylactic and therapeutic [82] antibody (mouse-humanized): s1 rbd hms-1 in vitro/in vivo (mouse) [83] antibody (human): s1 rbd in vitro/in vivo (mouse) potential for more mers-specific agents [61] of exon activity could be candidates for mers-cov and other coronavirus therapies [51] . as with other coronaviruses, the mers-cov s protein is critical to host cell receptor binding and cell entry, and is considered to have been under strong positive selection pressure when the virus was transmitted to humans [52, 53] . hence the s protein is a major target for potential anti-mers-cov therapies [53] . the s protein of mers-cov is composed of s1 and s2 subunits ( fig. 2 ) [53] . in common with other coronaviruses, entry into host cells depends on the s1 subunit, which contains a receptor-binding domain (rbd) comprising a core subdomain and a receptor-binding motif (rbm). the mers-cov rbm differs from that of sars-cov and dictates that mers-cov uses the dpp4 receptor, as opposed to the ace-2 receptor [20, 21] . the infection process is shown in fig. 2 . dpp4, which is widely expressed in tissues, including the lung and kidneys, is critical in the species tropism of mers-cov infection; bat, human, camel, non-human primate and swine cells, for example, are permissive for mers-cov infection, whereas mouse, hamster and ferret are not [54, 55] . host species restriction has been attributed to differences in five amino acids involved in dpp4-rbd binding, with glycosylation of the mouse dpp4 also identified as being important in the inhibition of mers-cov infection [54] [55] [56] . the human dpp4 receptor is therefore a potential target for mers-covspecific therapeutics, in particular anti-dpp4 mabs (fig. 2 , table 1 ) [53] [54] [55] [56] . adenosine deaminase (ada), which is a dpp4-binding protein, competes with mers-cov for dpp4 binding and hence is a natural mers-cov antagonist; this gives potential insights for the development of therapeutic antagonists [55] . mers-cov binds to the permissive host cell dpp4 via the rbd of the s1 domain, one of the major targets for potential mers-cov therapies [53] . similarly to other coronaviruses, mers-cov then uses the s2 subunit for virus-host membrane fusion (fig. 2) . fusion results in cleavage of the s protein at the s1/s2 boundary by host proteases [57] . the s2 subunit contains the fusion peptide, two heptad repeat domains termed hr1 and hr2, and a transmembrane (tm) domain ( fig. 2 ) [57] . membrane fusion requires conformational rearrangement of s2, the formation of a sixhelix bundle (6hb) fusion core, of which hr1 and hr2 are essential elements, and exposure of the fusion peptide, which inserts itself into the host cell membrane [52, 57, 58] . hr2-derived peptides have been identified as potentially effective anti-viral agents for treatment of mers-cov [52] ( fig. 2 ; table 1 ). the availability of host cell proteases is essential for mers-cov entry into cells [23, 53] . the host proteases responsible for s protein cleavage at the s1/s2 boundary include the serine protease tmprss2, endosomal cathepsins such as cathepsin l, and furin protease [23, 53, [59] [60] [61] . in vitro studies suggest that uncleaved mers pseudovirus can enter host cells by cathepsin l-dependent endocytosis, but that cleavage of virus during maturation by host proteases such as tmprss2 results in viral entry at neutral ph and the formation of massive syncytia [58] . host cell proteases are therefore potential molecular therapeutic targets for mers-cov prophylaxis and/or treatment (fig. 2 , table 1 ). the tmprss2 inhibitor camostat, for example, has been identified as a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [59] . following host cell entry, mers-cov pp1a and pp1ab are synthesized and then cleaved by two viral proteases, the main protease (mpro/3clpro) and the papain-like protease (plpro) (fig. 2) [57, 63] . thus viral proteases represent further potential molecular targets for therapy ( table 1) . the recently described mers-cov mpro crystal structure resembles other coronavirus mpro proteases [60] . the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg), can inhibit mers cov protease activity in vitro, as can the immunosuppressant drug mycophenolic acid [61] . however, caution is required, as the results of studies on marmosets have associated use of mycophenolate mofetil with fatal disease and high viral loads [36] . other mers-cov proteins involved in helping the virus to circumvent the immune system also present potential molecular targets (fig. 2) . for example, the accessory protein products of orf 4a, 4b and 5 are interferon (ifn) antagonists [62, 63] . the orf 4a protein both inhibits type i ifn production via cytoplasmic and nuclear mechanisms, and interferes with the ifn-mediated interferon-stimulated response element (isre) promoter element signalling pathways [62, 63] . this gives a molecular level rationale for the use of ifn as a therapeutic option in mers-cov treatment. mers-cov can also infect dendritic cells and macrophages [26, 64, 65] . endosomal uptake of mers-cov by dendritic cells following binding via dpp4 prompts these cells to produce abundant amounts of type i and iii ifns [65] . this gives context to the ifn antagonism exhibited by mers-cov accessory proteins. recently, mers-cov has also been shown to infect t cells, which are rich in dpp4, both in vitro and in marmoset spleen [66] . this results in t cell apoptosis and could contribute significantly to viral pathogenesis and further emphasizes the potential therapeutic utility of molecular targeting of dpp4 and/or the mers-cov s protein. both convalescent plasma containing virus-specific antibodies and the use of specific mabs provide options for targeting mers-cov infection at a molecular level. the use of convalescent plasma [or hyperimmune iv immunoglobulin (hvig) from the plasma of convalescent donors] for infectious disease treatments has a long history, including in the treatment of respiratory diseases [67] [68] [69] [70] . for influenza and sars-cov infection, early convalescent plasma treatment within 4-5 days of symptoms is associated with decreased viral load and reduction in mortality [67] [68] [69] [70] . however, for sars-cov the quality of studies has been inconsistent and the results have been inconclusive, with a lack of adequate clinical trials [69, 70] . according to the phe and isaric-who position paper, convalescent plasma (or high neutralizing antibody titre products) is indicated for the treatment of serious mers-cov infection [42] . one rct on 35 critically ill patients with h1n1 infection identified a significant reduction in viral load and mortality in patients who received hvig within 5 days of the onset of symptoms [68] . to date, no rcts have been completed on the use of convalescent plasma/hvig in mers-cov patients. in the light of results from sars and influenza patients, an ongoing clinical trial on the safety and efficacy of convalescent plasma treatment for critically ill mers-cov patients was initiated in may 2014 and is due to report in june 2017 [75; clinicaltrials.gov identifier: nct02190799]. this trial is being carried out in ksa. however, as is common for convalescent plasma therapies, the trial has been affected by logistical and technical issues, including the availability of sufficient donors [71, 72] . issues can also arise in the collection of convalescent plasma that has sufficient levels of mers-cov antibodies, particularly outside the middle east [22, 72] . while there are two case reports in which intravenous immunoglobulin (ivig) was used in treatment of mers-cov, it is uncertain as to whether this contributed to patient recovery [73, 74] . thus, while convalescent plasma is a promising potential therapy for mers-cov, the available clinical evidence is very limited and the results of the ongoing clinical trial will be vital in guiding any future use [71] . focused development of neutralizing monoclonal antibodies targeted against specific mers-cov proteins has meanwhile yielded promising in vitro and/or in vivo results. monoclonal antibodies: s1-dpp4 binding a number of mouse and human neutralizing mabs against the s1 region of mers-cov have been developed and tested in vitro and/or in animal models [52] . targeting of s protein for therapeutic purposes was recently comprehensively reviewed by du et al. [53] [52] . in particular, the s1 rbd is a popular target, as mabs directed against this region have the most potent neutralizing capacity. however, in terms of vaccine development, neutralizing antibodies raised by immunization with full-length s or s1 protein expression vectors may produce a more effective immunogenicity through the targeting of multiple epitopes and the reduction of the possibility of escape mutations [75] . nevertheless, many mouse and human mabs targeting the s1 rbd have given promising results in vitro and in mouse models [19] . (table 1) . a mouse monoclonal antibody, mersmab1, blocks mers pseudovirus cell entry in vitro by binding to the rbd and preventing s1 binding to dpp4 [76] . meanwhile, the human monoclonal antibodies m336, m337 and m338, which target overlapping epitopes in the rbd, all potently neutralize pseudovirus and live mers-cov in vitro [77] . significantly, intraperitoneal injection of m336 has also been shown to have both prophylactic and therapeutic protective effects against mers-cov infection in a wellestablished human dpp4 (hdpp4)-expressing transgenic mouse model, and in rabbits [78, 79] . other anti-rbd human antibodies, mers-4 and mers-27, which recognize distinct rbd regions and block binding to dpp4, likewise have potent in vitro neutralizing activity against pseudovirus and live virus infection, and they also act synergistically [80, 81] . mers-4 has anti-syncytia formation activity [80] . the crystal structure of mers-27 bound to the dpp4 receptor revealed two critical rbd residues [81] . the crystal structure of another anti-rbd antibody 4c2, which was raised in mice, has also been elucidated. this has allowed the identification of an epitope that partially overlaps the rbd receptor binding unit [82] . 4c2 was consequently humanized to give an antibody with prophylactic and therapeutic properties, shown by a reduction of mers-cov lung viral titres in an ad5-hcd26 (hdpp4) transgenic mouse model [82] . another humanized anti-rbd antibody, hms-1, similarly has potent in vivo protective properties against fatal mers-cov infection in a transgenic hdpp4 mouse model [83] . the human antibody lca60 targets both the n-terminal domain (ntd) and the rbd of the s1 region, and was isolated from b cells of a mers-cov-infected human donor before being used to rapidly establish a stable cho cell line that can be used to reliably produce clinical grade antibody [84] . this is a promising candidate for clinical development, given the antibody's potent prophylactic and therapeutic activities against mers-cov infection in ad5/hdpp4 transgenic mice and type i interferon receptor (ifnar)-ko mice [84] . the human anti-rbd antibody 3b11-n is another promising candidate that prophylactically reduces lung pathology in rhesus monkeys infected with mers-cov [85] . targeting the s1-dpp4 interaction from the host side through the development of anti-dpp4 (cd26) antibodies is another possible therapeutic option. the anti-cd26 antibodies 2f9, 1f7 and ys110 target the mers-cov entry into cells in vitro [86] . the 2f9 epitope maps close to the binding site of ada, a natural dpp4 binding protein and mers-cov antagonist, while the 1f7 and ys110 epitopes lie outside this region [55, 86] . thus, targeting of the s1-dpp4 interaction by use of mabs is a promising strategy for the clinical development of molecular therapeutics against mers-cov. another molecular approach involves targeting of the s2-mediated mers-cov-host cell fusion element of the mers-cov infection cycle by use of antiviral peptides. the role of hr1 and hr2 in viral fusion makes them potentially effective molecular therapeutic targets. this has been borne out by in vitro and in vivo results obtained using hr2 peptides (table 1 ). hr1 peptides are ineffective antivirals as they aggregate in physiological solutions [87] [88] [89] . a peptide named hr2p, which spans residues 1251-1286 of hr2, effectively inhibits viral replication and s proteinmediated cell fusion in vitro [87] . a hr2p analogue named hr2p-m2 is an even more potent fusion blocker in vitro, and inhibits mers cov-expressing pseudovirus infection [90] . hr2p-m2 interacts with an hr1 peptide to effectively block 6hb bundle formation. in vivo hr2p-m2 intranasal administration to ad5/hdpp4 transgenic mice protected them from mers-cov infection, as evidenced by the reduction of the lung viral titres by more than 1000-fold [90] . the addition of ifn-b along with hr2p-m2 enhanced the protective effect [90] . thus, s2 hr2 peptides have potential as mers-cov intranasal antiviral treatments. the s protein is also the focus of a number of candidate vaccines (table 1 ) [75, [91] [92] [93] . a fusion product combining truncated rbd and the fc portion of human igg can bind human dpp4 and inhibit mers-cov infection in an in vitro cell culture model [91] . importantly, this rbd-igg fusion product can induce a humoral response in mice vaccinated by subcutaneous injection, hence blocking rbd-dpp4 binding and inhibiting mers-cov infection [91] . further in vivo studies have indicated that intranasal administration to mice induces similar long-term igg humoral responses to those achieved with subcutaneous injection, but superior cellular immune responses and local mucosal responses in lungs [92, 93] . this suggests that this type of construct is both potentially effective and readily deliverable by intranasal means. use of an adjuvant, particularly mf59, significantly improves the humoral and t cell immunogenicity of the rbd s377-588-fc igg fusion construct in subcutaneously immunized mice [94] . the possibility of using the s1 rbd as a vaccine molecular target for a range of divergent mers-cov strains and escape mutants has also been explored recently [95] . the use of five recombinant rbds with mutations observed in different mers-cov outbreaks or in camel strains induced potent neutralizing antibody responses against several mers-cov pseudoviruses [95] . however, while the rbd of the s1 subunit is a logical and promising target for mers-cov vaccine development, the epitope scope is relatively limited and full-length s protein may be a preferable option [75] . technical difficulties in stably expressing abundant quantities of full-length s protein have presented a barrier. however, studies on delivery options, including the use of adjuvants and nanoparticles, may help in overcoming such issues. one study undertaken by novavax (gaithersburg, maryland, usa) showed that the inoculation of mice by intramuscular injection with fulllength s protein proprietary nanoparticles produced a relatively low neutralizing antibody response after 21 days [96] . however, the addition of the adjuvants alum or matrix m1 resulted in a robust and sustained anti-mers-cov neutralizing antibody response [96] . [98] . these viruses are expected to enter clinical trials as a proposed prophylactic mers-cov vaccine. likewise, intramuscular injection of ad5 or ad41 expressing full-length s protein induces both antigen-specific t cell and neutralizing antibody responses in mice [99] . finally, intraperitoneal injection of measles virus expressing either membraneanchored, full-length s protein or soluble s protein lacking the tm domain induces robust mers-cov antigen-specific neutralizing antibody and cytotoxic t lymphocyte in interferon-a/b receptor (ifnar)-deficient mice [100] . recently, an mva-based vaccine expressing s protein has been shown to induce mucosal immunity in mers-cov-infected dromedary camels, with a reduction in excreted virus and viral transcripts [101] . this has potential for veterinary use and the reduction of cross-species infection of humans by camels [101] . dna plasmids gls-5300 is a dna-plasmid vaccine that encodes mers-cov s protein (table 1 ) [102, 103] . it was co-developed by inovio, geneone life science, inc. and the walter reed army institute of research, and has become the first potential mers-cov vaccine to enter human testing [102, 103] . a phase i clinical trial in healthy volunteers commenced in 2016 for the evaluation of its safety and ability to generate antibody and cellular immune responses over a 1-year period, using one of three dosages in a three-injection regimen [102] . the vaccine has already undergone pre-clinical trials in mice, camels and macaques [103] . it induced robust and antigen-specific cytotoxic t lymphocyte and neutralizing antibody responses, which effectively protected animals against viral infection [103] . gls-5300 and other potential vaccine candidates provide an opportunity to develop a prophylactic mers-cov vaccine. however, the barriers to development of a prophylactic vaccine include the current relatively low mers-cov incidence in humans, as well as sourcing suitable small animal models [75, 97, 104] . these factors complicate the definition of a target population for mass prophylactic vaccination and pre-clinical demonstration of vaccine efficacy [104] . in this context, the monoclonal antibodies described above may be invaluable resources in an outbreak situation [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] . in vitro and animal studies while mers-cov-specific therapies are offering promising pre-clinical results, and gls-5300 has entered clinical trials, there is as yet no specific evidence-based therapy or vaccine clinically available for mers-cov. as described in the mers-cov infection and replication section, mers-cov accessory protein products are ifn antagonists [62, 63] . attenuation of the ifn response is an important mers-cov immune response circumvention mechanism [105] . the orf4a in particular inhibits ifn-b production via the inhibition of interferon regulatory transcription factor (irf)-3 and nuclear factor (nf)-kb actions, and thus irf-3-activating small molecules, for example, may be potential therapeutic agents for restoring ifn responses [62, 63] . toll-like receptor-3 (tlr-3) is also involved in the immune response of mice to sars-cov and mers-cov, recognizing viral molecular patterns and initiating the innate response that leads to ifn production (fig. 2 ) [106] . thus, tlr-3 agonists are another possible candidate for mers-cov-specific anti-viral agents [106] . therapeutically, ifn itself is particularly useful prophylactically or during the early days of viral exposure, including for coronaviruses [105, 107] . in vitro and animal studies have confirmed the potential efficacy of ifns in mers-cov therapy, in particular in combination with other therapeutic agents such as ribavirin and/or lopinavir. in vitro, mers-cov was substantially more susceptible to ifn-a than sars-cov [107] . while mers-cov in vero or llc-mk2 cells was sensitive to both ifn-a2b and ribavirin separately, relatively high concentrations were required to reduce viral replication [108] . however, combination therapy allowed the concentrations of each to be substantially reduced [108] . combination therapy of ifn-a2b and ribavirin in macaques administered 8 hours after mers-cov infection reduced systemic and local viral effects, and reduced viral genome copy number and gene expression levels [109] . bioinformatics data from microarray analysis recently showed that ifn-a2b and ribavirin treatment impacts on mers-cov gene expression in 10 different pathways, including genes involved in recognition of pathogens, immune responses and release of cytokines [110] . both ifn-b1b and lopinavir treatment, alone or in combination, also protected marmosets from the adverse clinical, radiological and pathological effects of mers-cov infection [111] . clinically, the use of ifn monotherapy, or ifn therapy in combination with ribavirin and/or lopinavir/ritonavir, has shown some promise (table 1 ) [37] [38] [39] [40] . however, the interpretation of clinical studies has been complicated by variability in factors such as the stage of infection at which therapy was administered. the available data are limited to case studies and retrospective cohort studies [22, 40] . in one case study on a patient who died in a greek hospital, pegylated ifn along with ribavirin and lopinavir was administered as part of the treatment regime, but not until the thirteenth day of the illness [39] . by contrast, in another preliminary study on two patients, the first patient was treated with ifn-a2b and ribavirin within a day of admission prior to mers-cov diagnosis, but he was also being treated with antibiotics, steroids and non-invasive ventilation [37] . patient 2, the wife of patient 1, was treated prophylactically after developing a low-grade fever and poorly defined lung infiltrates, but a diagnosis of mers-cov was not formally made [37] . thus, while patient 1 survived and patient 2 had only a mild course of illness, it is difficult to draw any firm conclusions regarding the efficacy of the treatment. in another case study on a patient in korea, administration of pegylated ifn-a2a along with ribavirin and lopinavir 4 days after hospital admission was deemed to have been effective in viral clearance and patient survival [38] . these case studies do not overall provide firm evidence for the efficacy or otherwise of ifn combination therapy for mers-cov. in one case involving a series of five patients who were critically ill with mers-cov infection and on mechanical ventilation and corticosteroids, ifn-a2b and ribavirin was administered on average 19 days after admission [27] . all five patients died, but they may not have benefited, as they were treated late in their illness and were already critically ill [27] . the benefit of earlier treatment in less vulnerable patients was suggested in another series of six patients in which three who received ifn-a2b and ribavirin early in the illness survived, while three other patients who were older and had comorbid conditions received the combination treatment later and all died [112] . however, in another study in which 20 mechanically ventilated patients with severe mers-cov infection who received pegylated ifn-a 2a and ribavirin early in treatment were compared to 24 patients who did not receive the combination therapy, the 14-day survival rate was significantly higher in the treatment group, but the 28-day survival rate was equivalently low in the two groups [113] . in another retrospective analysis of results from a series of 32 patients who received either ifn-a2a or ifn-b1a in combination with ribavirin, no significant difference in outcome between the two types of ifn was shown, and there was no survival benefit due to use of either ifn [29] . however, most of the patients in this study were aged more than 50 years and some had comorbid conditions, including end-stage renal disease [29] . thus the retrospective studies that have been carried out are heterogeneous in terms of type of patient, stage of disease and type of ifn used, including whether or not it was pegylated or short-acting. there is an urgent need for well-controlled clinical trials for ifn combination therapy in mers-cov, preferably early in the illness, as ifns are routinely available agents whose safety and efficacy is established for other viral illnesses and whose use has a sound molecular basis for mers-cov treatment. another type of therapy with a logical molecular basis for mers-cov treatment is the targeting of proteases, both host and viral (table 1 ; fig. 2 ) [19, 23, 53, 59, 60, [114] [115] [116] [117] . camostat, an inhibitor of tmprss2, is a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [59] . in a pathogenic mouse model of sars-cov infection, viral spread and pathogenesis was effectively blocked by camostat, and it is likely that it would have a similar impact on mers-cov [59] . as camostat is already in clinical use for the treatment of chronic pancreatitis, it represents a potentially safe and effective therapeutic option. recently, another tmprss2 inhibitor, nafamostat, was identified in a split protein-based cell-cell fusion assay as a potent inhibitor of mers-cov s protein-mediated hostviral membrane fusion in vitro [118] . nafamostat is already clinically approved for use by the us food and drug administration (fda) and is used as an anticoagulant [118] . the cathepsin l inhibitor teicoplanin, a glycopeptide antibiotic, was recently shown, via high throughput screening of fda-approved drugs, to block entry of mers-cov, sars-cov and ebola pseudoviruses into the cytoplasm [119] . teicoplanin is currently used clinically as an antibiotic in both prophylaxis and the treatment of serious grampositive bacterial infections. it also has derivatives, including dalbavancin, oritavancin and telavancin, all of which also block viral entry. the viral proteases, mpro (3clpro) and pl(pro), also represent potential molecular therapeutic targets [57, 60] . as well as its role in viral maturation, the mers-cov pl(pro) causes deubiquitination of ifn regulatory factor 3 (irf-3), and hence suppression of ifn b production, which contributes to viral suppression of the innate immune response (fig. 2) [120, 121] . the x-ray 3d crystal structure of mers-cov pl(pro) is similar to that of sars-cov, and includes ubiquitin-like and catalytic core domains [120] . thus the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg), can inhibit mers cov protease activity in vitro [61] . however, the mers-cov pl pro crystal structure also has unique aspects, including the oxyanion hole, and s3 and s5 subsites, which may be viable molecular targets for antivirals specifically designed against mers-cov [120] . a commercial compound termed compound 4 (commercial code f2124-0890,life chemicals) has been identified as an inhibitor of mers-cov and sars-cov plpro activity [122, 123] . the critical binding interactions and mode of inhibition differ between the two viral proteases, with the compound acting as a competitive inhibitor against mers-cov pl(pro), but an allosteric inhibitor of sars-cov pl[pro) [122] . however, f2124-0890 may lose potency in physiological reducing environments [123] . lopinavir is a protease inhibitor with activity against the sars-cov main protease m pro [124] . in a screen of a library of 348 fda-approved drugs to identify anti-mers-cov activity in cell culture, lopinavir emerged as one of four compounds that inhibited viral activity in a low micromolar range [125] . however, the clinical efficacy of lopinavir in mers-cov treatment has not yet been fully established. as mentioned above, it has usually been used clinically in combination with ifn and data are only available from case studies and series. however, notably, lopinavir-ritonavir treatment resulted in better clinical, radiological and pathological outcomes and reduced mortality in marmosets infected with mers-cov [36] . lopinavir has also been identified in a position paper from phe and isaric-who as a potential mers-cov therapy whose benefits are likely to exceed its risks [47] . thus far, mers-cov has not been considered to have pandemic potential. most cases have occurred in the middle east, particularly in ksa. outbreaks have been primarily linked to healthcare institutions, and shortcomings in infection control and prevention procedures [6] [7] [8] [9] [10] [11] [12] [13] [14] . however, potential viral mutations could facilitate expanded viral host range and enhance cross-species and human-human transmission [20, 58, 114] . the outbreak in korea resulted in mers-cov emergence in second-and third-generation contacts, highlighting the potential for mutational changes that could increase the likelihood of human-human transmission [14, 18] . mers-cov also exacts a high mortality rate, mainly due to the development of ards [15] [16] [17] . these factors emphasize the importance of developing targeted therapies and/or vaccines. the most promising advances in the development of specific molecular mers-cov therapies relate to targeting of the viral s protein by means of anti-s1monoclonal antibodies, hr-targeted antiviral peptides and viruses or plasmids bearing s protein as potential vaccine candidates [52, 55, 58, [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] . the use of ifns, usually in combination with other therapies such as ribavirin or lopinavir, also has a logical molecular basis given that ifn antagonism is an important mechanism by which the virus circumvents the innate immune system [62, 63, 105, 107] . targeting of host and viral proteases is also a sound molecular approach, as host proteases are important in viral-host membrane fusion, while viral proteases are key to viral maturation and are also involved in targeting ifns [23, 53, [59] [60] [61] [114] [115] [116] [117] . the therapies currently used for mers-cov have mainly been extrapolated from those used for sars-cov treatment, regardless of the important differences in receptor usage and cellular tropism between the viruses [20] [21] [22] [23] [24] [25] [26] . none of these therapies have been subject to well-controlled trials, and in some cases the risks are likely to outweigh any poorly defined benefits [19, 22, [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . in general, the clinical research response to mers-cov may have been too slow [126] . thus, while there are many promising lines of research in terms of specific molecular targeting of mers-cov, no potential therapies have yet been subject to welldesigned clinical trials, and none have been approved for clinical use, apart from the gls-5300 dna-plasmid vaccine [102, 105] . continuing outbreaks of mers-cov, with possible increases in human-human transmission, are likely to galvanize the research community to push ahead with the design and performance of clinical trials for some of the available monoclonal antibodies and/or antiviral peptides for use in outbreak situations. there are various challenges inherent in the development of specific mers-cov therapies. these include the difficulty of identifying a target population for potential prophylactic vaccines, limited small animal model availability and dependence on transgenic mouse models, and the current relatively low incidence of infection, which complicates the performance of adequate clinical trials [75, 96, 97, 104] . for example, one currently ongoing trial on convalescent plasma therapy has been affected by logistical and technical issues, including insufficient available donors and difficulty in collecting convalescent plasma containing sufficient mers-cov antibody levels [71, 72] . thus, while numerous monoclonal antibodies have been raised with anti-mers activity, in particular against the s protein [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] , and promising antiviral hr2 peptides have been synthesized [87, 90] , the available data are thus far limited to in vitro and animal studies. despite these issues, there is cause for optimism, given the many candidate antibody and peptide therapies. there is also some promising in vitro and animal model evidence suggesting that use of ifns, which are well-established therapies in other viral illnesses, may be of benefit if used sufficiently early in mers-cov treatment, or as a prophylactic, especially in combination with other therapies, including ribavirin or lopinavir-ritonavir [108] [109] [110] [111] . likewise, other drugs that are currently in clinical use for other conditions have been shown to be potentially useful for mers-cov treatment, including camostat and nafamostat; teicoplanin and its derivatives dalbavancin, oritavancin and telavancin; and the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg) [59, 61, [118] [119] [120] [121] . these drugs have already been shown to be safe and well-tolerated by humans. repurposing of existing drugs may therefore prove to be the most viable option in mers-cov therapy. for example, 1 screen of 290 approved drugs uncovered 27 candidates with in vitro activity against both mers-cov and sars-cov, including oestrogen receptor inhibitors and dopamine receptor inhibitors [127] . thus, there are many options available on a molecular level for the development of new mers-cov-specific therapies, as well as the adoption of drugs that are currently in use for other purposes, which should assist in more effective and reliable prevention and treatment of this virus. isolation of a novel coronavirus from a man with pneumonia in saudi arabia ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum 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infection mediated by the transmembrane serine protease tmprss2 human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) -possible lessons from a systematic review of sars-cov therapy ifn-a2a or ifn-b1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans prolonged disturbances of in vitro cytokine production in patients with severe acute respiratory syndrome (sars) treated with ribavirin and steroids cytokine responses in porcine respiratory coronavirus-infected pigs treated with corticosteroids as a model for severe acute respiratory syndrome effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients corticosteroid treatment in critically ill patients with pandemic influenza a/ h1n1 2009 infection: analytic strategy using propensity scores treatment with lopinavir/ritonavir or interferon-b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset ribavirin and interferon-a2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases combination therapy with lopinavir/ritonavir, ribavirin and interferon-a for middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen sensitivity of sars/mers cov to interferons and other drugs based on achievable serum concentrations in humans update on therapeutic options for middle east respiratory syndrome coronavirus treatment of mers-cov; information for clinicians. clinical decision-making support for treatment of mers-cov patients. www.google.ie/?gws_rd= ssl#q=public+health+england+treatment+mers-cov virus taxonomy: classification and nomenclature of viruses. ninth report of the international committee on taxonomy of viruses cross host transmission in the emergence of mers coronavirus rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a 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antibody protects rabbits from mers-cov infection potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection rapid generation of a human monoclonal antibody to combat middle east respiratory syndrome 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeatderived peptides protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection a truncated receptorbinding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines receptor-binding domain-based subunit vaccines against mers-cov identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice toward developing a preventive mers-cov vaccine-report from a workshop organized by the saudi arabia ministry of health and the international vaccine institute protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels gls-5300 syncon® immunotherapy targeting middle east respiratory syndrome. www.inovio.com/products/ infectious-disease-vaccines/mers a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates mers-cov vaccine candidates in development: the current landscape delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-a treatment inhibition of novel b coronavirus replication by a combination of interferon-a2b and ribavirin treatment with interferon-a2b and ribavirin improves outcome in mers-cov-infected rhesus macaques bioinformatics analysis on molecular mechanism of ribavirin and interferon-a in treating mers-cov treatment with lopinavir/ritonavir or interferon-b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein the heptad repeat region is a major selection target in mers-cov and related coronaviruses identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov x-ray structure and enzymatic activity profile of a core papain-like protease of mers coronavirus with utility for structure-based drug design small molecules targeting severe acute respiratory syndrome human coronavirus screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture towards improving clinical management of middle east respiratory syndrome coronavirus infection repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection mechanisms of coronavirus cell entry mediated by the viral spike protein the authors received no specific grant from any funding agency. the authors declare that there are no conflicts of interest. this is a review article and no experimental work with humans was performed. five reasons to publish your next article with a microbiology society journal 1. the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer reviewaverage time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-319877-izn315hb authors: de wit, emmie; van doremalen, neeltje; falzarano, darryl; munster, vincent j. title: sars and mers: recent insights into emerging coronaviruses date: 2016-06-27 journal: nat rev microbiol doi: 10.1038/nrmicro.2016.81 sha: doc_id: 319877 cord_uid: izn315hb the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. the continuing introductions of mers-cov from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. scientific advancements since the 2002–2003 severe acute respiratory syndrome coronavirus (sars-cov) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of mers-cov and the development of therapeutics. in this review, we detail our present understanding of the transmission and pathogenesis of sars-cov and mers-cov, and discuss the current state of development of measures to combat emerging coronaviruses. supplementary information: the online version of this article (doi:10.1038/nrmicro.2016.81) contains supplementary material, which is available to authorized users. mers 12 , and a cluster of three cases of mers in the uk was identified in september 2012 (ref. 13 ). mers-cov continued to emerge and spread to countries outside of the arabian peninsula as a result of travel of infected persons; often, these imported mers cases resulted in nosocomial transmission. in may 2015, a single person returning from the middle east started a nosocomial outbreak of mers in south korea that involved 16 hospitals and 186 patients 14 . as of 26 april 2016, there have been 1,728 confirmed cases of mers, including 624 deaths in 27 countries 15 . this review highlights the pandemic and epidemic potential of emerging coronaviruses and discusses our current knowledge of the biology of sars-cov and mers-cov, including their transmission, their pathogenesis and the development of medical countermeasures. key features of these viruses are the dominance of nosocomial transmission, and pathogenesis that is driven by a combination of viral replication in the lower respiratory tract and an aberrant host immune response. several potential treatments for sars and mers have been identified in animal and in vitro models, including small-molecule protease inhibitors, neutralizing antibodies and inhibitors of the host immune response. however, efficacy data from human clinical trials are lacking but are needed to move these potential countermeasures forward. respectively (fig. 1a) . similarly to all viruses in the order nidovirales, sars-cov and mers-cov have a unique coding strategy: two-thirds of the viral rna is translated into two large polyproteins, and the remainder of the viral genome is transcribed into a nested set of subgenomic mrnas 16, 17 (fig. 1b) . the two polyproteins, pp1a and pp1ab, encode 16 non-structural proteins (nsp1-nsp16) 18 that make up the viral replicase-transcriptase complex. the polyproteins are cleaved by two proteases, papainlike protease (plpro; corresponding to nsp3) and a main protease, 3c-like protease (3clpro; corresponding to nsp5). the nsps rearrange membranes that are derived from the rough endoplasmic reticulum (rer) into double-membrane vesicles, in which viral replication and transcription occur 19 . one unique feature of coronaviruses is the exoribonuclease (exon) function of nsp14 (ref. 20) , which provides the proofreading capability required to maintain a large rna genome without the accumulation of detrimental mutations 21, 22 . sars-cov and mers-cov transcribe 12 and 9 subgenomic rnas, er-golgi intermediate compartment (ergic) . a cellular compartment that facilitates transport between the endoplasmic reticulum (er) and the golgi complex. infected individuals who each infect a disproportionately large number of secondary cases. respectively, and these encode the four structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as several accessory proteins that are not involved in viral replication but interfere with the host innate immune response or are of unknown or poorly understood function. the envelope spike glycoprotein binds to its cellular receptor, angiotensin-converting enzyme 2 (ace2) for sars-cov and dipeptidyl peptidase 4 (dpp4) for mers-cov 23 . after membrane fusion, either directly with the host cell membrane or with the endosome membrane, the viral rna genome is released into the cytoplasm, and the rna is uncoated to allow translation of the two polyproteins, transcription of the subgenomic rnas and replication of the viral genome (fig. 1b) . newly formed envelope glycoproteins are inserted in the rer or golgi membranes; genomic rna and nucleocapsid proteins combine to form nucleocapsids, and the viral particles bud into the er-golgi intermediate compartment (ergic). virion-containing vesicles subsequently fuse with the plasma membrane to release the virus 24 . the first indication of the source of sars-cov was the detection of the virus in masked palm civets and a raccoon dog and the detection of antibodies against the virus in chinese ferret badgers in a live-animal market in shenzhen, china 25 . however, these animals were only incidental hosts, as there was no evidence for the circulation of sars-cov-like viruses in palm civets in the wild or in breeding facilities 26 . rather, bats are the reservoir of a wide variety of coronaviruses, including sars-cov-like and mers-cov-like viruses 27 (fig. 2) . thus, the search for the reservoir of mers-cov initially focused on bats, but a serological survey in dromedary camels from oman and the canary islands showed a high prevalence of mers-cov-neutralizing antibodies in these animals 28 . in addition, mers-cov rna was detected in swabs that were collected from dromedary camels at a farm in qatar that was linked to two human cases of mers, and infectious virus was isolated from dromedary camels in saudi arabia and qatar [29] [30] [31] [32] . serological evidence for the circulation of a mers-cov-like virus in dromedary camels has been obtained in the middle east, eastern africa and northern africa, dating back as far as 1983 (ref. 33 ). dromedary camels in saudi arabia harbour several viral genetic lineages 34 , including those that have caused human outbreaks. taken together, these data strongly point to the role of dromedary camels as a reservoir for mers-cov. the ubiquity of infected dromedary camels close to humans and the resulting continuing zoonotic transmission may explain why mers-cov continues to cause infections in humans, whereas sars-cov, without the continuing presence of an infected intermediate host and with relatively infrequent human-bat interactions, has caused no more infections in humans. human-to-human transmission of sars-cov and mers-cov occurs mainly through nosocomial transmission; 43.5-100% of mers cases in individual outbreaks were linked to hospitals, and very similar observations were made for some of the sars clusters 35, 36 . transmission between family members occurred in only 13-21% of mers cases and 22-39% of sars cases. transmission of mers-cov between patients was the most common route of infection (62-79% of cases), whereas for sars-cov, infection of health care workers by infected patients was very frequent (33-42%) 35 . the predominance of nosocomial transmission is probably due to the fact that substantial virus shedding occurs only after the onset of symptoms 37, 38 , when most patients are already seeking medical care 39 . an analysis of hospital surfaces after the treatment of patients with mers showed the ubiquitous presence of viral rna in the environment for several days after patients no longer tested positive 40 . moreover, many patients with sars or mers were infected through super spreaders 14, 35, 37, [41] [42] [43] . the clinical courses of sars and mers are remarkably similar, although there are subtle differences (box 1) . owing to the current sparsity of data on human mers-cov infections 44 , the pathogenesis of this virus is poorly understood; however, similar mechanisms may underlie the pathogenesis of both mers and sars. the binding of spike protein to ace2 and the subsequent downregulation of this receptor contribute to lung injury during sars 45 . although it seems counterintuitive that receptor downregulation would increase pathology, it has been shown that ace2 can protect against acute lung injury. the downregulation of ace2 results in the excessive production of angiotensin ii by the related enzyme ace, and it has been suggested that the stimulation of type 1a angiotensin ii receptor and middle east respiratory syndrome coronavirus (mers-cov) encode two large polyproteins, pp1a and pp1ab, which are proteolytically cleaved into 16 non-structural proteins (nsps), including papain-like protease (plpro), 3c-like protease (3clpro), rna-dependent rna polymerase (rdrp), helicase (hel) and exonuclease (exon). an additional 9-12 orfs are encoded through the transcription of a nested set of subgenomic rnas. sars-cov and mers-cov form spherical particles that consist of four structural proteins. the envelope glycoprotein spike (s) forms a layer of glycoproteins that protrude from the envelope. two additional transmembrane glycoproteins are incorporated in the virion: envelope (e) and membrane (m). inside the viral envelope resides the helical nucleocapsid, which consists of the viral positive-sense rna ((+)rna) genome encapsidated by protein nucleocapsid (n). b | following entry of the virus into the host cell, the viral rna is uncoated in the cytoplasm. orf1a and orf1ab are translated to produce pp1a and pp1ab, which are cleaved by the proteases that are encoded by orf1a to yield 16 nsps that form the rna replicase-transcriptase complex. this complex localizes to modified intracellular membranes that are derived from the rough endoplasmic reticulum (er) in the perinuclear region, and it drives the production of negative-sense rnas ((-)rnas) through both replication and transcription. during replication, full-length (-)rna copies of the genome are produced and used as templates for full-length (+)rna genomes. during transcription, a subset of 7-9 subgenomic rnas, including those encoding all structural proteins, is produced through discontinuous transcription. in this process, subgenomic (-)rnas are synthesized by combining varying lengths of the 3′end of the genome with the 5′ leader sequence necessary for translation. these subgenomic (-)rnas are then transcribed into subgenomic (+)mrnas. although the different subgenomic mrnas may contain several orfs, only the first orf (that closest to the 5′end) is translated. the resulting structural proteins are assembled into the nucleocapsid and viral envelope at the er-golgi intermediate compartment (ergic), followed by release of the nascent virion from the infected cell. one of a panel of recombinant inbred mouse strains derived from a genetically diverse set of founder strains and designed for the analysis of complex traits. the aggregation of leukocytes around blood vessels. (agtr1a) increases pulmonary vascular permeability, thus potentially explaining the increased lung pathology when the expression of ace2 is decreased 46 . immunopathology. the immune response is essential for the resolution of an infection, but it can also result in immunopathogenesis. one indication that immunopathogenesis may contribute to sars was the observation that viral loads were found to be decreasing while disease severity increased 39, 47 . it is unclear whether a similar trend applies to mers 48, 49 . moreover, progression to acute respiratory distress syndrome (ards) is associated with the upregulation of pro-inflammatory cytokines and chemokines, particularly interleukin-1β (il-1β), il-8, il-6, cxc-chemokine ligand 10 (cxcl10) and cc-chemokine ligand 2 (ccl2) 50, 51 ; increased plasma levels of these molecules have been detected in patients with sars [52] [53] [54] [55] . retrospective longitudinal studies in patients who recovered from sars versus those who succumbed to the disease have shown an early expression of interferon-α (ifnα), ifnγ, cxcl10, ccl2 and proteins that are encoded by ifn-stimulated genes (isgs) in all patients, but only patients who survived then had gene expression profiles that are indicative of the development of an adaptive immune response. by contrast, patients who succumbed maintained high levels of cxcl10, ccl2 and isg-encoded proteins, whereas spike-specific antibodies were present at low levels or were absent 56 , which suggests that severe disease is related to the lack of a switch from an innate immune response to an adaptive immune response. experiments using collaborative cross mouse lines and mouse-adapted sars-cov identified one host gene, trim55, as important for sars pathogenesis. although there was no difference in clinical signs or viral replication in trim55 −/− mice compared with wild-type mice, perivascular cuffing and the number of inflammatory cells in the lungs were reduced in the trim55 −/− mice 57 . a biological process in which small rna molecules induce the degradation of specific mrna molecules, thereby inhibiting gene expression. systems in which a dna molecule is produced that contains the viral leader and trailer sequences, with an assayable reporter replacing the viral orfs. when combined with the expression of viral proteins in trans, this system can be used to model the viral life cycle without the necessity of using infectious virus. the involvement of the host immune response in the pathogenesis of sars, and most likely also that of mers, suggests that drugs which inhibit viral replication will need to be combined with treatments that control detrimental immune responses. immune evasion. sars-cov and mers-cov use several strategies to avoid the innate immune response. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna (dsrna) or uncapped mrna, are detected by pattern recognition receptors (prrs), such as retinoic acid-inducible gene i protein (rig-i; also known as ddx58) or melanoma differentiation-associated protein 5 (mda5; also known as ifih1) 58 . this triggers complex signalling cascades involving myd88 that lead to the production of type i ifns and the activation of the transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induces the transcription of pro-inflammatory cytokines (fig. 3a) . type i ifns signal through ifnα/β receptor (ifnar) and downstream molecules such as signal transducer and activator of transcription (stat) proteins to stimulate the production of antiviral proteins that are encoded by isgs, such as ifn-induced protein with tetratricopeptide repeats 1 (ifit1 ; fig. 3b ). collectively, this establishes an antiviral immune response that limits viral replication in infected and in neighbouring cells (fig. 3) . infection of knockout mice revealed the importance of innate immunity. infection of myd88 −/− and stat1 −/− mice, but not mice that were deficient in ifn receptors, with a mouse-adapted strain of sars-cov resulted in more severe disease than infection with a non-adapted sars-cov strain 59, 60 . moreover, mers-cov infection of wild-type mice that were transduced with human dpp4 caused mild disease, but symptoms were more severe in myd88 −/− mice and ifnar1 −/− mice 61 . sars-cov and mers-cov avoid host detection of their dsrna by replicating in virus-induced doublemembrane vesicles that lack prrs 19, 62, 63 . moreover, the recognition of sars-cov mrnas, for example, by mda5 and ifit1 is prevented by capping of the viral mrnas by nsp14 and the nsp10-nsp16 complex 64 . recombinant sars-cov that lacks the methylation activity of nsp16 is attenuated and exhibits increased sensitivity to type i ifns. this effect is dependent on ifit1 or mda5, as the same virus is not attenuated in mice that are deficient in either molecule 65 . although mrna capping has not yet been shown for mers-cov, structural similarity between the mers-cov nsp10-nsp16 complex and the sars-cov nsp10-nsp16 complex suggests that a similar mechanism exists to avoid host recognition of mers-cov mrnas by cytosolic prrs 66 . sars-cov encodes at least eight proteins that interact with the signalling cascades downstream of prrs; in mers-cov, several proteins have been identified with similar functions (fig. 3) . the nucleocapsid protein of sars-cov has been associated with the suppression of rnai in mammalian cells 67 . furthermore, this protein antagonizes ifn induction, probably early in the signalling cascade, as downstream signalling molecules relieve the inhibition 68 . mers-cov orf4a has a similar ifn-antagonistic function, involving the binding of dsrna and subsequent inhibition of mda5 activation 69 , potentially through interaction with ifn-inducible dsrna-dependent protein kinase activator a (prkra; also known as pact), which interacts with mda5 and rig-i 70 . moreover, mers-cov orf4a, orf4b, orf5 and membrane protein inhibit the nuclear trafficking of ifn-regulatory factor 3 (irf3) and activation of the ifnb promoter 71 . these viral proteins, except for orf5, also inhibit the expression of genes that are under the control of an ifn-stimulated response element (isre), and orf4a reduces the expression of genes that are stimulated by nf-κb 71 . finally, mers-cov orf4b interacts with tbk1 and inhibitor of nf-κb kinase-ε (ikkε), thereby suppressing the interaction between ikkε and mitochondrial antiviral-signalling protein (mavs) and inhibiting the phosphorylation of irf3 (ref. 72 ). the membrane protein of sars-cov inhibits the formation of a signalling complex that contains ikkε, thus repressing the activation of irf3 and irf7 and their induction of type i ifn expression. the membrane protein of mers-cov inhibits irf3 function and the expression of genes that are regulated by an isre, including ifnβ 71 , but whether this occurs through a mechanism similar to that of sars-cov is unclear. sars-cov plpro disrupts nf-κb signalling 73 and blocks the phosphorylation of irf3 indirectly 73, 74 . furthermore, sars-cov plpro inhibits the induction of type i ifns, potentially through the deubiquitylation of phosphorylated irf3 (refs 73, 75) . similar functions have been described for mers-cov plpro 76 . experiments involving recombinantly expressed proteins, in vitro translation, protein overexpression and minireplicon systems have shown that nsp1 of sars-cov blocks the ifn response through the inhibition of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have an incubation period of ~5 days, and 95% of patients develop disease within 13 days of exposure 14, 38, [144] [145] [146] . common early symptoms are fever, chills, coughing, malaise, myalgia and headache, and less common symptoms include diarrhoea, vomiting and nausea 2, 6, 39, 89, 90, 92, 95, 144, [146] [147] [148] . upper respiratory tract symptoms and viral shedding are rare, which explains difficulties in obtaining a laboratory diagnosis from nasal or nasopharyngeal swabs 149 . abnormal chest x-rays are more common in patients with mers (90-100%) 144,148 than in those with sars (60-100%) 39, 89 . accordingly, only 20-30% of patients with sars require intensive care and subsequent mechanical ventilation, whereas 50-89% of patients with mers require intensive care 2, 39, 89, 90, 95, 144, 147, 148 . the higher incidence of acute respiratory distress syndrome (ards) in individuals with mers is reflected in the case fatality rate: this is ~36% for mers compared with ~10% for sars 15, 145 . comorbidities have an important role in both sars and mers. several risk factors are associated with poor disease outcome, especially advanced age and male sex 2, 14, 39, 144, 146, 148, 150, 151 . for mers, additional risk factors for a poor outcome include diabetes mellitus, hypertension, cancer, renal and lung disease, and co-infections 14, 144, 146, 148, 150, 151 . health care settings seem to increase the risk of viral transmission owing to aerosol-generating procedures such as intubation and bronchoscopy. appropriate hospital hygiene practices and awareness are crucial to limit future nosocomial outbreaks. both nsp7 and nsp15 from sars-cov were also suggested to be ifn antagonists, but the underlying mechanism is unknown 73 . nsp15 is an inhibitor of mavsinduced apoptosis; however, this occurs through an ifn-independent mechanism 84 . finally, transcriptomic and proteomic analysis of human airway cell cultures showed that mers-cov but not sars-cov induces repressive histone modifications that downregulate the expression of certain isgs 85 . it should be noted that most of the interactions of sars-cov and mers-cov proteins with innate immune pathways were established in in vitro systems, which rely on the overexpression of viral and, . following prr-mediated detection of a pamp, the resulting interaction of prrs with mitochondrial antiviral-signalling protein (mavs) activates nuclear factor-κb (nf-κb) through a signalling cascade involving several kinases. activated nf-κb translocates to the nucleus, where it induces the transcription of pro-inflammatory cytokines. the kinases also phosphorylate (p) ifn-regulatory factor 3 (irf3) and irf7, which form homodimers and heterodimers and enter the nucleus to initiate the transcription of type i interferons (type i ifns). both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have developed mechanisms to interfere with these signalling pathways, as shown; these subversion strategies involve both structural proteins (membrane (m) and nucleocapsid (n)) and non-structural proteins (nsp1, nsp3b, nsp4a, nsp4b, nsp5, nsp6 and papain-like protease (plpro); indicated in the figure by just their nsp numbers and letters). b | binding of type i ifns to their dimeric receptor, ifnα/β receptor (ifnar), activates the janus kinase (jak)-signal transducer and activator of transcription (stat) signalling pathway, in which jak1 and tyk2 kinases phosphorylate stat1 and stat2, which form complexes with irf9. these complexes move into the nucleus to initiate the transcription of ifn-stimulated genes (isgs) under the control of promoters that contain an ifn-stimulated response element (isre). collectively, the expression of cytokines, ifns and isgs establishes an antiviral innate immune response that limits viral replication in infected and in neighbouring cells. again, viral proteins have been shown to inhibit these host signalling pathways to evade this immune response. iκbα, nf-κb inhibitor-α. a broadly active antiviral nucleoside analogue with several direct and indirect mechanisms of action; mainly used for the treatment of hepatitis c, in combination with interferon. having polyethylene glycol (peg) attached, to a drug for example; this moiety improves the solubility, decreases the immunogenicity and increases the stability, of the drug of interest, thereby allowing a reduced dosing frequency to be used. (table 1) and sars-cov, including the use of antibodies, ifns, inhibitors of viral and host proteases, and host-directed therapies. in the absence of a clinically proven effective antiviral therapy against sars-cov and mers-cov, patients mainly receive supportive care, which is often supplemented by different combinations of drugs. ribavirin 86 and various types of ifn have been given to patients with mers in saudi arabia 87 and china 88 , typically in combination with a broad-spectrum antibiotic and oxygen. the efficacy of treatments for sars-cov and mers-cov infection currently remains unclear. in addition, treatment for mers is typically started only in a late disease stage, when immunopathology predominates and antiviral drugs are likely to provide little benefit. ribavirin was used most frequently during the sars outbreak, often in combination with corticosteroids, which have an anti-inflammatory effect 2,89-92 . ifnα was also given, usually in combination with immunoglobulins or thymosins, which stimulate the development of t cells, and in a small number of cases in combination with ribavirin 93, 94 . none of these treatments was tested in a clinical trial, which makes it difficult to assess their efficacy. in fact, retrospective analysis did not yield a treatment combination that was clearly effective. moreover, the data from patients are contradictory about whether ribavirin, when used alone, provided a benefit or was possibly even detrimental 89, 90, 92, 95 . in vitro coronaviruses have a lower sensitivity to ribavirin than other viruses. deletion of the nsp14-encoding sequence increases the sensitivity of coronaviruses to ribavirin; however, the underlying mechanism is unclear and is not related to the proofreading function of nsp14 (ref. 96 ). therefore, ribavirin should be considered only in combination with other antiviral treatments. although ifns are effective against mers-cov in vitro [97] [98] [99] , their effect in humans has yet to be proved. the effectiveness of ifn is increased in vitro if ribavirin is added 98, 100 , and a combined use of the two drugs reduces disease severity in a rhesus macaque model of mers 101 . the potential side effects of these treatments, such as fatigue, depression and anaemia, have inhibited their use as a first-line treatment for mers, and they are generally administered only after a patient's condition starts to deteriorate. for example, one study of five patients who were infected with mers-cov indicated no survival following ribavirin and ifnα2b therapy; however, therapy was not started until 10 days after admission 87 . a separate study found an improvement in survival 14 days after mers diagnosis and the start of treatment, but not 28 days after 102 . in a third study, a combination of ifnα2a and ribavirin or ifnβ1a and ribavirin did not improve survival; however, some of the patients were more than 50 years old and had preexisting renal failure 103 . in a single case in which ribavirin and ifnα2b were started shortly after admission to hospital, the patient started to improve on day 6 after admission and made a complete recovery 104 . ifnβ1b is a more potent inhibitor of mers-cov replication in vitro than other types of ifn 97, 99 , and an improved outcome of disease was observed in common marmosets after challenge with mers-cov 105 . thus, the type of ifn that is used for treatment in humans should be reconsidered (usually, ifnα is used). furthermore, ribavirin and/or ifns should be tested in clinical trials to determine their efficacy in mers treatment and to establish treatment protocols. additional antiviral treatments. the protease inhibitors lopinavir and ritonavir, which are used in combination to treat infection with hiv, improved the outcome of patients with sars when combined with ribavirin, compared with patients who were treated with ribavirin alone 106, 107 . lopinavir showed no clear antiviral activity against mers-cov in vitro 97 , and it is thus rarely used in patients with mers. however, lopinavir and ritonavir improve the outcome in common marmosets when treatment is initiated 6 hours after infection with mers-cov 105 . thus, the testing of lopinavir and ritonavir in clinical trials in patients with mers should be reconsidered. one patient who received pegylated ifnα, ribavirin, lopinavir and ritonavir in combination had undetectable levels of mers-cov in the blood 2 days after the initiation of therapy; however, this patient did not survive 108 . the combination of ifnα, ribavirin, lopinavir and ritonavir was also used for mers treatment in south korea, but efficacy data are not yet available. however, three case reports indicate recovery in five out of seven patients who were treated with this combination [109] [110] [111] . as 3clpro and plpro are essential for cleavage of the viral polyproteins and are distinct from cellular proteases, they are ideal drug targets, in particular plpro, compounds that mimic biologically active peptides or proteins. a complement-activated molecule that is important for the recruitment to and activation of inflammatory cells in the lungs. which is involved in both viral replication and ifn antagonism. indeed, most antiviral drug-like molecules have been developed against 3clpro and plpro, which was aided by the rapid report of crystal structures of these proteases 112 . plpro was initially identified as a drugable target for sars-cov; recently, it has been noted that some of the compounds that target plpro from sars-cov are also active against plpro from mers-cov. for example, both 6-mercaptopurine and 6-thioguanine inhibit mers-cov and sars-cov in vitro 113 ; however, the efficacy of these molecules has not yet been tested in vivo. mycophenolic acid also inhibits the replication of mers-cov in vitro 97, 99 through the inhibition of plpro 113 , but it had no effect in marmosets 105 . as new coronaviruses are likely to emerge from bats, protease inhibitors were designed against bat coronaviruses with the goal of developing a universal antiviral compound against emerging zoonotic coronaviruses. this approach yielded an inhibitor of tylonycteris bat coronavirus hku4 (hku4-cov), which is closely related to mers-cov 11 . this inhibitor, named sg85, indeed inhibits mers-cov replication in vitro 112 . similarly, peptidomimetics that target and inhibit 3clpro of mers-cov, hku4-cov and pipistrellus bat coronavirus hku5 (hku5-cov) have also been identified, but have not yet progressed beyond the in vitro stage 114 . several other drugs that were approved for use in humans were shown to inhibit the replication of mers-cov in vitro, notably chloroquine, chlorpromazine, loperamide and cyclosporine a 62, 99, 113, 115 , although their mechanisms of action are unknown, and the benefit of cyclosporine a in patients is debatable owing to the immunosuppressive effect of the drug. although cyclophilin inhibitors that do not result in immunosuppression are available, their activity against mers-cov has not yet been tested. antibody and plasma therapy. plasma from convalescent patients and/or antibody therapies have been the leading proposed treatment for mers so far 116 . there are several potential advantages to this approach. for example, as case numbers increase, the pool of survivors becomes larger; provided these individuals have sufficiently high antibody titres and are willing and able to donate plasma, this is a low-tech, reasonably safe treatment option. furthermore, generation of monoclonal antibodies for use in humans is well established, with a fairly straightforward path to safety and efficacy testing. however, to date, there are very few reports on the use of convalescent plasma and none on the use of monoclonal antibodies as treatments for acute, severe respiratory disease in humans. a post hoc meta-analysis of 32 studies of either sars or severe influenza found a significant reduction in the pooled odds of mortality when convalescent plasma was used 117 . however, study design was rated as low or very low quality, as there were generally a lack of control groups and a moderate-to-high risk of bias, which suggests that a properly designed clinical trial of convalescent plasma use in severe respiratory infections is needed 117 . potent monoclonal antibodies that neutralize the mers-cov spike protein in vitro have been developed [118] [119] [120] [121] . however, with a few exceptions, in vivo data relating to the use of convalescent plasma or monoclonal antibodies in the treatment of mers are currently lacking. serum from high-titre dromedary camels decreased mers-cov loads in the lungs of mice that had been transduced with human dpp4 (ref. 122 ). human polyclonal antibodies against the spike protein were generated by vaccinating transchromosomic bovines, and treatment with these antibodies reduced viral titres in the lungs of dpp4-transduced mice when treatment was administered 24 or 48 hours after challenge with mers-cov 123 . dpp4-transduced mice were also treated with humanized neutralizing monoclonal antibody 4c2h, which is directed against the receptor-binding domain of the mers-cov spike protein, 1 day after mers-cov challenge, and this treatment also decreased viral titres in the lungs 124 , as did the neutralizing antibody lca60, which was obtained from a convalescent patient and produced recombinantly 125 . human neutralizing monoclonal antibodies regn3048 and regn3051 also provided a benefit in mice that expressed human dpp4 and were challenged with mers-cov 126 . the human neutralizing monoclonal antibody m332 reduced mers-cov replication in the lungs of rabbits following prophylactic, but not therapeutic, treatment 127 . treatment of rhesus macaques with the human monoclonal antibody 311b-n1 day after challenge resulted in reduced lung pathology 128 . in all of these studies, viral replication was not completely inhibited, and there were some pathological alterations to the lungs, despite the therapy. furthermore, none of the studies addressed the potential emergence of escape mutants in vivo. alternatively, antibodies that target the region of dpp4 that binds to the spike protein could be used to prevent entry of mers-cov; this approach was successful in vitro 129 . however, whether such a treatment would be feasible and would not have substantial adverse effects in humans remains to be determined. host-directed strategies can also limit viral replication. for example, the spike protein of sars-cov is cleaved by cathepsin b and cathepsin l, transmembrane protease serine 2 (tmprss2) and possibly other host proteases 130, 131 . inhibition of host serine proteases by camostat reduced the entry of sars-cov and increased survival in a mouse model 132 . however, the targeting of host proteases is more likely to result in undesirable side effects than the targeting of viral proteases. another underappreciated strategy is attenuation of detrimental host responses. the development of such treatments would require a thorough understanding of the host responses that are involved in acute lung injury and ards, processes that are unfortunately poorly understood at the moment. nonetheless, in vitro studies and limited studies in animal models with other respiratory viruses have shown that anaphylatoxin c5a is important for the development of acute lung injury, and blocking anaphylatoxin c5a can reduce lung pathology 133 . vaccines that contain immunogenic parts of a pathogen rather than the entire pathogen. vaccines based on the direct introduction of a plasmid encoding an antigen; following in situ production of this antigen, an immune response is mounted against it. changes in gene expression during in vitro mers-cov infection were used to predict potential effective drugs. one of the drugs with predicted efficacy, the kinase inhibitor sb203580, modestly inhibited sars-cov and mers-cov replication following the treatment of cells prior to infection, but treatment after infection inhibited replication of only sars-cov and not mers-cov 134 . vaccines. vaccination could be used to prevent infection or to reduce disease severity, viral shedding and thereby transmission, thus helping to control mers outbreaks. several vaccination strategies were developed against sars-cov and tested in animals, such as an inactivated virus, a live-attenuated virus, viral vectors, subunit vaccines, recombinant proteins and dna vaccines 135, 136 . similar approaches have been used for the development of experimental mers-cov vaccines 137 . to date, three mers-cov vaccines have been evaluated in non-human primates. in one study, rhesus macaques were primed with dna encoding the spike protein, followed by boosts with spike dna and with recombinant protein consisting of the spike subunit containing the receptor-binding domain, or primed and boosted once with the subunit protein. both approaches reduced pathological changes in lung function in animals that were infected with mers-cov 19 weeks after the last vaccination 138 . moreover, three vaccinations with a recombinantly expressed protein that contains the receptor-binding domain of the spike protein reduced viral loads and lung pathology in rhesus macaques that were infected 2 weeks after the last vaccination 139 . three dna vaccinations with a construct encoding the full-length spike sequence reduced viral loads and pathology in the lungs after challenge with mers-cov 5 weeks after the last vaccination 140 . one concern of vaccination in humans is vaccinemediated enhancement of disease, a process in which the disease following infection is more severe in vaccinated individuals than in unvaccinated individuals. although this was observed in only a small subset of vaccine studies that were carried out for sars-cov 136 and has not yet been observed in any of the published mers-cov vaccine studies, it is an important concern. moreover, it is unclear who to vaccinate against mers-cov, as healthy individuals seem to be at little risk of severe disease. older patients or patients with underlying disease, who have the highest risk of severe mers, would be important target populations. however, vaccination in such patients can be problematic owing to their poor immune responses, as has been established for influenza virus 141 . in addition, vaccination of people with a high risk of exposure to mers-cov, such as health care workers, slaughterhouse workers and camel herders, is advisable 142 . as our understanding of the pathogenesis of emerging coronaviruses increases, so will the opportunities for the rational design of therapeutics that target viral replication or immunopathology. the rational design of new drugs and the repurposing of existing compounds have already resulted in the development of plpro inhibitors and the identification of kinase inhibitors that inhibit the replication of sars-cov and mers-cov in vitro. however, only a few potential treatments have progressed past the identification of an effect in vitro, and in vivo studies to select the most promising treatment options are required. the development of several mouse models of mers is thus an important step forward (box 2) . owing to the acute nature of mers and the important role of immunopathology, combination therapies aimed at simultaneously inhibiting viral replication, limiting viral dissemination and dampening the host response are likely to yield the best results. furthermore, treatment should be started as early as possible, rather than waiting until the patient has already developed extensive lung damage. the development of therapies against sars and mers needs to focus on testing in humans, in properly controlled clinical trials. the current non-standardized, uncontrolled approach to treatment is not informative and may not be beneficial to the patient. the recent ebola outbreak has demonstrated that rapid clinical trial design and approval are possible and that exceptional situations call for deviations from normal procedures . while treatments are being developed and evaluated, the prevention of viral transmission is key to reducing the burden of mers. the large proportion of nosocomial mers-cov infections indicates that preventive measures in hospitals are currently either not fully implemented or insufficient. prevention of zoonotic transmission from dromedary camels is another possibility to decrease the number of mers cases. the most of our understanding of the pathogenesis of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) comes from animal studies. ideally, these models recapitulate all or specific aspects of human disease. several mouse models have been established, for example by using mouse-adapted sars coronavirus (sars-cov) or expressing human receptors in mice 152 . although it has been recognized that mice might poorly mimic specific human responses to infection, the availability of knockout and transgenic mice enables the targeted study of virus-host interactions. several non-human primate models have been developed for sars-cov and mers coronavirus (mers-cov) 152 . the close relationship of non-human primates to humans often allows faithful recapitulation of a disease and the host response. however, these benefits are countered by the need for specialized husbandry, the sometimes limited availability of the animals and reagents, and high costs. the pathogenesis of sars-cov and mers-cov in their respective reservoir hosts is not nearly as well studied as that in humans. currently, only one experimental-infection study has been carried out in bats with mers-cov 153 , and none has been carried out for other coronaviruses. thus, data are mostly limited to the detection of coronaviruses in naturally infected bats. the detection of coronaviruses mainly in faecal samples from bats and not in oral swabs suggests that replication in bats occurs predominantly in the gastrointestinal tract 9,154,155 . by contrast, a combination of intratracheal and intranasal inoculation of masked palm civets with sars-cov resulted in interstitial pneumonia, with oral and rectal viral shedding 156 . the pathogenesis of mers-cov in dromedary camels has been studied experimentally in a limited number of animals. these animals developed transient mild disease; however, large quantities of mers-cov were shed from the upper respiratory tract, in line with the predominant replication of mers-cov in the nasal turbinates and larynx in these animals, which explains the frequent zoonotic transmission 157 . first vaccines against mers-cov have been tested in dromedary camels 140, 143 ; indeed, when camels were vaccinated with a modified vaccinia virus that expresses the mers-cov spike protein, subsequent challenge of these animals with mers-cov resulted in less viral shedding than in unvaccinated animals 143 , thereby potentially limiting the transmission to naive animals or to humans. certainly, there has been progress in the development of vaccines and therapies against emerging coronaviruses, but more research and rigorous testing is required if we are to successfully combat these novel pathogens. when the severe acute respiratory syndrome (sars) outbreak developed into the first pandemic of the twenty-first century, it became clear that the medical and scientific communities were not adequately prepared for the emergence of highly pathogenic viruses. whereas several months elapsed and several thousand cases of sars were observed before the causative agent was identified as sars coronavirus (sars-cov) 4-6 , subsequent advances in molecular diagnostic tools, such as next generation sequencing, meant that middle east respiratory syndrome coronavirus (mers-cov) was identified before it caused a large outbreak of mers 11 . the availability of the full-length genome of mers-cov enabled the rapid development and distribution of diagnostic assays. the first animal models of disease, several treatment efficacy studies and the identification of the reservoir followed soon after. unfortunately, the sars pandemic did not yield solid clinical data on the efficacy of treatment regimens. these data are urgently needed for the treatment of mers, as well as to prepare for novel coronaviruses that may emerge. several studies have used synthetic biology to study the zoonotic transmission potential of sars-cov-like viruses from bats 9,10,158,159 . the ebola virus outbreak in west africa has highlighted the need for fast-tracking of potential treatments, as several clinical trials were started only towards the end of the outbreak. the combined experiences of the outbreaks of sars, mers and ebola provide a blueprint for the response to emerging pathogens: after the identification of the causative agent, diagnostic assays need to be developed and distributed rapidly, and simultaneously, awareness of the new syndrome and reporting of (suspected) cases 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syndrome coronavirus isolates the first description of mers-cov replication and shedding in the respiratory tract of dromedary camels synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice sars-like wiv1-cov poised for human emergence the work was supported by the intramural research program of the national institute of allergy and infectious diseases (niaid), us national institutes of health. the authors declare no competing interests. key: cord-022582-2e9i3m4b authors: potsic, william p.; wetmore, ralph f. title: otolaryngologic disorders date: 2012-03-21 journal: pediatric surgery doi: 10.1016/b978-0-323-02842-4.50055-3 sha: doc_id: 22582 cord_uid: 2e9i3m4b nan the ear is divided into three anatomic and functional areas: the external ear, the middle ear, and the inner ear. the external ear consists of the auricle, external auditory canal, and the lateral surface of the tympanic membrane. the auricle is a complex fibroelastic skeleton that is covered by skin and subcutaneous tissue that directs sound into the external ear canal. the external auditory canal is oval with the long axis in the superior to inferior direction. in neonates, the external canal is almost entirely supported by soft, collapsible cartilage. as the temporal bone grows over several years, the bony portion of the canal enlarges to comprise the inner one third, leaving the outer two thirds supported by firm cartilage. hair and cerumen glands are present in the outer two thirds of the external canal. the ear canal is lined by skin that is continuous with the lateral surface of the tympanic membrane, and it is innervated by cranial nerves v, vii, and x and cervical nerve iii. the tympanic membrane separates the external ear canal from the middle ear. it has three layers: an outer layer of skin; a middle layer of fibrous tissue that is attached to the malleus, the most lateral middle ear ossicle; and an inner layer of mucosa that is continuous with the mucosa lining the middle ear. the fibrous layer is also attached to a thick fibrous annulus that anchors it to the temporal bone. the middle ear is an air-filled space within the temporal bone of the skull that is lined by ciliated, columnar respiratory epithelium. the middle ear communicates with the mastoid air cell system posteriorly and is lined by the same mucosa. it also communicates with the nasopharynx anteriorly through the eustachian tube. the mucociliary transport system of the middle ear moves mucus and debris into the nasopharynx, where it is swallowed. secretory cells are not evenly distributed throughout the middle ear and mastoid complex and are more numerous anteriorly near the eustachian tube. three ossicles are present in the middle ear-the malleus, incus, and stapes-that transmit sound from the vibrating tympanic membrane to the stapes footplate. stapes movement creates a fluid wave in the inner ear that travels to the round window membrane and is dissipated by reciprocal motion to the stapes. there are two striated muscles in the middle ear. the tensor tympani muscle lies along the side of the eustachian tube, and its tendon attaches to the medial surface of the malleus. the stapedius muscle lies along the vertical portion of the facial nerve in the posterosuperior part of the middle ear. its tendon attaches to the head of the stapes. these muscles stiffen the ossicular chain in the presence of sustained loud noise. the facial nerve traverses the middle ear with its horizontal portion lying superior to the stapes. posterior to the stapes, the facial nerve turns inferiorly in a vertical fashion to exit the stylomastoid foramen deep to the tip of the mastoid. the chorda tympani nerve is a branch of the facial nerve that innervates taste to the anterior two thirds of the tongue. it exits the facial nerve in the vertical segment and passes under the posterosuperior surface of the tympanic membrane, crossing the middle ear lateral to the long process of the incus and medial to the malleus. the facial nerve lies within a protective bony canal throughout its course in the middle ear. however, the bony canal may be absent (in the horizontal portion) in as many as 30% of patients. cranial nerve ix supplies sensation to the floor of the middle ear. the inner ear consists of the cochlea, semicircular canals, and vestibule. the cochlea is a coiled fluid-filled tube consisting of 2 3 / 4 turns surrounded by dense bone. it contains the membranes that support the organ of corti and has hair cells that detect the fluid wave from vibration of the stapes footplate. the hair cells create the neural impulses that are transmitted from the auditory nerve (cranial nerve viii) to the brain, providing the sensation of hearing. the three paired semicircular canals (horizontal, superior, and inferior) are also fluid-filled tubes surrounded by dense bone. the semicircular canals each have a hair cell-containing structure (the ampulla) that detects motion. the utricle and saccule of the vestibule also have hair cell structures that detect acceleration. 26 the external ear develops during the sixth week of gestation and is completely developed by the 20th week. six hillocks fuse to form the basic units of the pinna. defects in the fusion of the hillocks lead to preauricular tags and sinuses. the external auditory canal develops from the first pharyngeal cleft. a solid epithelial plug forms during the beginning of the third month of gestation and canalizes in the seventh month to form the external auditory canal. the middle ear canal develops from the first pharyngeal pouch. the ossicles develop from the first and second pharyngeal arches. the inner ear arises from neuroectodermal tissue within the otic placode that forms the otic pit. 26 any combination of anomalies may occur. abnormalities of the development of the ear may create anomalies of the pinna, external auditory canal, middle ear structures, and inner ear. one of the anomalies that involves the external and middle ear is aural atresia (absence of the external auditory canal). absence of the external canal may occur with a deformed or normal external ear. the ossicles may be deformed and are usually fused to each other as well as the bony plate representing the undeveloped tympanic membrane. the facial nerve may also be altered in its course through the temporal bone. reconstruction of the atretic canal, removal of the bony tympanic plate, release of the fused ossicles, and reconstruction of a new eardrum is a complex surgical procedure that may improve hearing. rarely there is incomplete development of the inner ear structures. the most common of these is dysplasia of the cochlea, and it may vary in severity. dysplasia causes sensorineural hearing loss. 6, 14 the examination of the ear should always start with inspection of the outer ear and surrounding structures. deformities of the outer ear structure may suggest the presence of other anomalies, such as a first branchial cleft sinus. a first branchial cleft sinus usually presents below the ear lobe near the angle of the jaw. the sinus tract may connect to the ear canal or, rarely, the middle ear. the external auditory canal and tympanic membrane are best examined with a hand-held otoscope with a bright fiberoptic light source. the largest speculum that comfortably fits in the external canal should be used to maximize visualization and minimize pain. a very small speculum may be inserted deeply, but it might lacerate the ear canal as well as limit visibility of the tympanic membrane. the otoscope permits visualization of the ear canal and tympanic membrane. a translucent tympanic membrane will also permit visualization of the contents of the middle ear. cerumen may be encountered in the ear canal that obstructs the view of the tympanic membrane. removal of cerumen may be performed by using an operating otoscope head and an ear curet. however, the use of a headlight such as the lumiview (welch allyn, skaneateles, ny) or operating microscope permits the use of both hands and superior visualization. care should be taken to secure the child to prevent sudden movement, and the ear curet should be used gently to avoid causing pain and a laceration of the ear canal. examination of a child with an apparent or suspected ear condition often requires objective assessment of hearing by audiometry. current technology and expertise makes it possible to test a child at any age. behavioral audiometry can usually be accurately performed for a child who is older than 6 months of age by sound-field testing. older children are presented with a tone through insert earphones and a range of frequencies between 250 and 8000 hz for ear-specific testing. the hearing thresholds are recorded at each presented frequency; and this represents the air conduction threshold. the sound has to traverse the ear canal, tympanic membrane, and middle ear. the inner ear must respond by creating electrical impulses that are transmitted to the brain. normal thresholds are less than 20 db for children. bone conduction thresholds test the sensorineural component of hearing. a bone oscillator is used to test a range of frequencies by vibrating the skull, which stimulates the inner ear, directly bypassing the external and middle ear. normally, air conduction thresholds require less energy than bone conduction thresholds. if bone conduction thresholds require less sound intensity than air conduction to be heard, the child has a conductive hearing loss. if air conduction and bone conduction thresholds are elevated but the same, the child has a sensorineural hearing loss. most sensorineural hearing loss in children is a result of hair cell dysfunction in the organ of corti. hearing loss may be conductive, sensorineural, or mixed. electrophysical tests such as brainstem auditory evoked response and sound emission tests that measure the intrinsic sounds from the inner ear (otoacoustic emissions) may be employed in young infants and children who cannot participate in behavioral audiometry. a mechanical test of tympanic membrane compliance (tympanometry) may also be used for audiometric assessment. all of these tools are employed by pediatric audiologists. 23 for purposes of describing hearing loss, a threshold between 20 and 40 db is considered mild, 40 to 65 db is moderate, 55 to 70 db is moderately severe, 70 to 90 db is severe, and greater than 90 db is profound. four of 1000 children are born with a hearing loss, and 1 of those children is born with a severe to profound hearing loss. conductive hearing loss may be corrected with otologic surgery. hearing aids and fm systems may be helpful to children with both conductive and sensorineural hearing loss. assistance may be needed through auditory training, speech language therapy, and education to maximally develop communication skills. when a child has a sensorineural hearing loss that is too severe to be helped with hearing aids, a cochlear implant may be considered. a cochlear implant is an electrical device that is implanted under the scalp behind the ear. its processor converts sound to electrical impulses. a cable travels through the mastoid and facial recess to reach the middle ear, and the electrode array is inserted into the scala tympani of the cochlea through an opening that is made in the cochlea. cochlear implants stimulate the neural elements of the cochlea directly and bypass the hair cells. because the vast majority of sensorineural hearing loss in children is due to hair cell dysfunction, nearly all children get sound perception from a cochlear implant. rare conditions such as an absent auditory nerve or an absent cochlea preclude the use of a cochlear implant. a multidisciplinary evaluation by a cochlear implant team is required to evaluate a child and determine family expectations before performing a cochlear implant. a temporal bone computed tomographic (ct) scan and/or magnetic resonance imaging (mri) is performed to assess the cochlea and auditory nerves. children who are born deaf and are younger than the age of 3 years, as well as children who have already developed communication skills, language, and speech before losing their hearing, derive the greatest benefit from cochlear implants. cochlear implantation is approved for children 12 months of age or older by the u.s. food and drug administration. after a cochlear implant is performed, considerable auditory oral training is required to maximize a child's benefit to develop skills of audition, speech, and language. a child who has been deaf and without sound perception for several years is expected to benefit to a lesser degree. 33 otitis media with effusion is the most common chronic condition of the ear during childhood. all children are born with small eustachian tubes that may at times be unable to clear mucus that is secreted in the mastoid and middle ear. fluid may develop in the middle ear during an upper respiratory infection. it usually clears within a few weeks as the upper respiratory tract infection resolves. children with craniofacial anomalies such as cleft palate and down syndrome are also prone to middle ear effusions; there is no medication that is consistently effective in resolving such effusions. persistent effusion may cause a conductive hearing loss in the range of 20 to 40 db. a middle ear effusion may also function as a culture medium and predispose children to recurrent acute suppurative otitis media (asom). when fluid persists in the middle ear for 3 to 4 months, causing a hearing loss or is associated with asom, myringotomy and tympanostomy tube placement is helpful to resolve the hearing loss and reduce the frequency and severity of infection. myringotomy and placement of a tube is performed under general anesthesia using an operating microscope. a small incision is made in any quadrant of the tympanic membrane except the posterosuperior quadrant, where there would be risk of injuring the ossicles. the mucus is suctioned from the ear, and a silastic tube is placed in the myringotomy to provide prolonged ventilation of the middle ear. the tube will usually be extruded and the tympanostomy will heal in 6 months to 1 year. when the ear is no longer ventilated by a tube, the eustachian tube must ventilate the middle ear. if fluid recurs and persists, a repeat procedure may be needed. most children outgrow this problem as their eustachian tube grows. occasionally, adenoid tissue in the nasopharynx may contribute to the persistence of middle ear effusion and may also be removed at the time that a tube is placed. children who have had multiple sets of tubes are candidates for adenoidectomy. acute suppurative otitis media is the most common infection of childhood except for acute upper respiratory tract infections. it is the most common condition for which children seek medical care from their primary care physician. usual pathogens causing asom include streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis. 25 acute suppurative otitis media usually causes severe deep ear pain, fever, and a conductive hearing loss in the affected ear. the purulence in the middle ear is also present in the mastoid air cells because they are connected. asom is treated with broad-spectrum oral antibiotics; however, there is growing concern that indiscriminant use of antibiotics may result in antibiotic resistance. for this reason, accurate diagnosis by otoscopy should be made before initiating a course of antibiotics. occasionally, asom does not respond as expected to standard antibiotic therapy. when this occurs, culture and sensitivity testing can be obtained by tympanocentesis. after sterilizing the ear canal with alcohol, a 22-gauge spinal needle can be placed through the posterior or anterior inferior quadrant of the tympanic membrane and fluid can be aspirated with a small syringe. complications of asom are uncommon if appropriate antibiotic therapy is used. the conductive hearing loss resolves as the middle ear effusion clears. however, infection may necrose the tympanic membrane, causing a spontaneous perforation. small perforations usually heal in less than 7 days, but larger perforations may persist, cause a conductive hearing loss, and require a tympanoplasty for closure. the ossicular chain may also be disrupted by necrosis of the long process of the incus requiring ossicular reconstruction to restore hearing. acute coalescent mastoiditis occurs when infection erodes the bony mastoid cortex and destroys bony septa within the mastoid. a subperiosteal abscess may also be present. there is usually postauricular erythema and edema over the mastoid area. the auricle is displaced laterally and forward ( fig. 52-1 ). otoscopy reveals forward displacement of the posterior superior skin of the ear canal. in addition to antibiotics, treatment should include a wide field myringotomy from the anterior inferior quadrant to the posterior inferior quadrant, a tympanostomy tube placement for middle ear drainage, and a postauricular mastoidectomy to drain the subperiosteal abscess and the mastoid. facial nerve paralysis may occur from inflammation of that portion of the facial nerve that is exposed in the otolaryngologic disorders middle ear during asom. treatment with parenteral antibiotics, ototopical antibiotic drops applied in the ear canal, and a wide field myringotomy and tympanostomy tube placement almost always result in complete recovery of facial function. facial nerve recovery may take a few weeks to several months. intracranial complications of asom may include meningitis, epidural abscess, brain abscess, otitic hydrocephalus, and lateral sinus thrombosis. meningitis is the most common intracranial complication of asom and may be associated with profound sensorineural hearing loss and loss of vestibular function. treatment of the intracranial complications of asom is focused on appropriate treatment of the intracranial process, in addition to a wide field myringotomy and tympanostomy tube placement in the affected ear. 2 chronic otitis media is a descriptive term that refers to a persistent perforation of the tympanic membrane or the presence of a cholesteatoma of the middle ear. a cholesteatoma is a squamous epithelial-lined cyst that may be congenital or acquired. congenital cholesteatomas are caused by epithelial rests that persist in the middle ear during temporal bone development. they present behind an intact tympanic membrane and appear as a white, smooth mass in the middle ear. they expand over time and are filled with squamous debris and may erode the ossicular chain and extend into the mastoid. acquired cholesteatoma develops from skin entering the middle ear after a tympanic membrane perforation or a retraction pocket from eustachian tube dysfunction. cholesteatomas are usually painless, cause a conductive hearing loss, and, in acquired cases, often present as otorrhea. the otorrhea should be treated with ototopical antibiotic eardrops, but the only treatment of cholesteatomas is complete surgical excision by tympanomastoid surgery and ossicular reconstruction. 27[pp 18-59] the potential complications of cholesteatomas are the same as those for asom. objects stuck deeply into the ear canal such as a cottontipped applicator may perforate the tympanic membrane. this usually causes acute pain, bleeding, and a conductive hearing loss. if the ossicular chain is not disrupted, the vast majority of these perforations will heal spontaneously in about 2 weeks. if the tympanic membrane is perforated and the middle ear is contaminated with water, oral antibiotics should be given. lacerations of the auricle should be cleaned to prevent tattooing and repaired by careful approximation of the skin and soft tissue to restore the contours of the ear. the cartilage itself does not usually need to be sutured. partially or totally avulsed tissue should be replaced. if necrosis of tissue occurs, it can be dã©brided as needed. in severe injuries of the auricle, oral antibiotic treatment is helpful to prevent chondritis and loss of the cartilage framework. blunt trauma to the ear is commonly seen in wrestlers, in children with poor neuromuscular tone, or in children with self-injurious behaviors. blood or serum collects between the periosteum and the auricular cartilage. if the cartilage is fractured, the collection may occur on both sides of the ear. evacuation of the collection is required to restore the contours of the ear, prevent infection, and prevent scarring with formation of a "cauliflower ear." aspiration of the fluid and placement of a mastoid dressing for compression may be tried but is most often unsuccessful. incision and drainage provides for complete evacuation of the blood or serum. cotton dental rolls placed in each side of the auricle and held in place with bolster mattress sutures is the most effective management. the dental rolls should be left in place for 7 to 10 days while the patient also continues with a course of oral antibiotics. no outer dressing is required. 27[pp 106-109] blunt head trauma may disrupt the inner ear membranes causing sensorineural hearing loss and vertigo. no treatment is required, and the injury and symptoms may resolve spontaneously, but the sensorineural hearing loss may persist. severe head trauma may cause fracture of the temporal bone of the skull. temporal bone fractures can be classified as longitudinal, transverse, or mixed ( fig. 52-2 ) but are often complex and do not neatly fit into one category or another. a high-resolution, thin section ct scan of the temporal bone will define the extent of the fracture. the middle ear and mastoid are filled with blood when a fracture is present. the blood causes a conductive hearing loss that resolves when the ear clears. otoscopic evaluation of a child with a temporal bone fracture may reveal a laceration of the ear canal and tympanic membrane. blood is usually present in the ear canal, and the tympanic membrane appears to be dark blue because the middle ear is filled with blood. there is often ecchymosis of the mastoid area (battle's sign). it is important during evaluation of a skull and temporal bone fracture to note and record the function of the facial nerve if the patient is not unconscious. facial nerve paralysis may be immediate or delayed in onset. delayed facial nerve paralysis has a good prognosis for spontaneous recovery. immediate facial paralysis may indicate disruption of the nerve or compression by bone fragments. immediate facial nerve paralysis requires exploration and repair once the patient is stable and sufficiently recovered from any associated trauma. the facial nerve should be decompressed in the mastoid, middle ear, and middle cranial fossa. bone chips impinging on the nerve should be removed, and the nerve should be sutured or grafted if needed. all patients with temporal bone fractures should have an audiogram once their condition has stabilized. if the fracture disarticulates the ossicles, a conductive hearing loss will persist after the blood has cleared from the middle ear and mastoid. fractures of the temporal bone may transverse the cochlea and vestibular apparatus. these fractures usually cause a severe sensorineural hearing loss and loss of vestibular function on the affected side. a concussive injury of the cochlea may also simultaneously be present in the opposite ear in severe head trauma. temporal bone fractures may permit leakage of cerebrospinal fluid (csf) into the middle ear and mastoid. csf may also drain through the lacerated tympanic membrane, causing csf otorrhea. these leaks usually stop spontaneously, but persistent csf otorrhea may require a lumbar drain to reduce the pressure and permit healing. rarely, tympanomastoid exploration is required to close the leak. persistent csf leaks in the ear are associated with meningitis. benign and malignant tumors of the ear are rare. glomus tympanicum tumors and neuromas of the facial nerve may present in the middle ear. also, eosinophilic granuloma and rhabdomyosarcoma may involve the structures of the temporal bone. 5, 13 the nose can be divided into three anatomic sections. the bony vault is the immobile portion of the nose. it consists of the paired nasal bones, the frontal process of the maxillary bone, and the nasal process of the frontal bone. the cartilaginous vault is supported by the upper lateral cartilages and the cartilaginous nasal septum. the nasal lobule is supported by the lower lateral cartilages and the cartilaginous septum. the nasal septum is formed by the quadrilateral cartilage anteriorly. the posterior septum is composed of bone from the vomer, perpendicular plate of the ethmoid, nasal crest of the maxillary bone, and palatine bone. both the internal and external carotid artery systems supply blood to the nose. the roof and lateral wall of the internal nasal cavity are supplied by the anterior and posterior ethmoidal arteries, sphenopalatine artery, and greater palatine artery. the septum is supplied by the anterior and posterior ethmoidal arteries, palatine artery, and the superior labial artery. the convergence of these vessels in the anterior segment of the nose is referred to as kiesselbach's plexus or little's area. venous drainage is accomplished mainly by the ophthalmic, anterior facial, and sphenopalatine veins. the olfactory bulb is positioned high in the roof of the nasal cavity and is responsible for the sense of smell. sensory information is transported by nerves that penetrate the cribriform plate and traverse cranial nerve i (the olfactory nerve) to the brain. smell is also an important component of what is perceived as taste. bony projections, turbinates, form the lateral nasal wall and significantly increase the surface area of the nose, allowing for more efficient humidification and warming of the air to 36â°c. three turbinates are usually present (i.e., inferior, middle, and superior). a supreme turbinate, which is essentially a flap of mucosa, is occasionally present. the turbinates contribute to the turbulent airflow that creates approximately 50% of the total airflow resistance to the lungs. cleaning of air is accomplished through the nasal hairs (vibrissae) and the mucosal surface. anteriorly, the nose is lined with stratified squamous epithelium, which changes to respiratory epithelium immediately anterior to the turbinates. trapped debris is transported in a posterior direction into the nasopharynx by a mucociliary transport mechanism. speech is affected by nasal anatomy and pathologic conditions. hyponasality from nasal obstruction or hypernasality from an excessive air leak can affect voice quality and intelligibility of speech. the nose serves as a drainage port for the paranasal sinuses. the meati are spaces between the lateral aspect of the nasal turbinates and the medial aspects of the lateral nasal wall. each meatus is named for the turbinate that surrounds it. the maxillary, frontal, and anterior ethmoidal sinuses drain into the middle meatus. the posterior ethmoidal sinuses drain into the superior meatus. the sphenoidal sinus drains into an area known as the sphenoethmoidal recess that is located posterior and superior to the superior turbinate. the nasolacrimal duct drains into the inferior meatus. the nasal cavities develop from the nasal pits in the 4-week embryo. these pits deepen and move medially to form the nasal cavity. the oronasal membrane that separates the nose from the mouth resolves in the seventh week to permit communication between the nose and nasopharynx. the paranasal sinuses develop from an outpouching of the lateral nasal walls during the third and fourth months of development. the maxillary and ethmoidal sinuses are present at birth. the frontal and sphenoidal sinuses begin to develop several years after birth. the frontal sinus begins to develop at 7 years of age but is not fully aerated until adulthood. 1 viral rhinosinusitis (the common cold) accounts for the majority of nose and sinus infections. it is caused by many strains of viruses and is a self-limited infection. symptoms of fever, nasal congestion, headache, and clear rhinorrhea usually resolve over 5 to 7 days. treatment is symptomatic. acute bacterial rhinosinusitis may often follow an acute viral upper respiratory tract infection. the most common bacteria causing rhinosinusitis are streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis. acute rhinosinusitis causes malaise, headache, and nasal congestion. there may also be pain localized to the sinus region or pain on palpation over the maxillary or frontal sinuses. chronic sinus infection may persist after the acute phase and symptoms often last longer than 30 days. the "gold standard" for diagnosing sinusitis is a ct of the sinuses, but a thorough history and nasal examination is usually sufficient to diagnose acute rhinosinusitis. the nasal cavity can be visualized by using a large speculum on an otoscopic head. the posterior nasal cavity can be visualized with either a straight rod endoscope or a flexible fiberoptic nasopharyngoscope. the treatment of rhinosinusitis includes oral antibiotics, short-term use of topical nasal decongestants (e.g., oxymethazoline), and saline nasal sprays. topical nasal corticosteroid sprays may be helpful for the treatment of chronic sinusitis. chronic sinusitis in a child may be exacerbated by gastroesophageal reflux disease, immunodeficiencies, mucociliary dysfunction, and, more commonly, upper respiratory allergy. these predisposing conditions should be managed while treating the sinus infection. if the signs and symptoms of chronic sinus infection persist, a sinus ct is required to evaluate the condition of the sinus mucosa and the drainage pathways. endoscopic sinus surgery may be necessary to open the involved sinuses to provide drainage. chronic inflammation of the nasal and sinus mucosa may lead to nasal and sinus polyp formation that chronically obstructs the nose and sinuses. antrochoanal polyps are large polyps that originate from the walls of the maxillary sinus and extend through the nasal cavity into the nasopharynx. nasal polyps may be removed endoscopically, but a large antrochoanal polyp may require removal through an open maxillary sinus procedure. nasal polyps in a child should always prompt an evaluation for cystic fibrosis. the sinuses surround the orbit so a common complication of acute rhinosinusitis in children is orbital cellulitis with erythema and edema of the eyelids. chemosis (edema of the ocular conjunctiva) is usually absent. however, if a periorbital subperiosteal abscess forms adjacent to an infected sinus, there may be proptosis, chemosis, ophthalmoplegia, and loss of vision. infection in the ethmoidal sinuses most commonly results in this complication. subperiosteal periorbital abscess is demonstrated best by sinus ct. initial treatment should include intravenous antibiotics. endoscopic or external drainage may be required in some cases. intracranial complications of sinusitis include cerebritis, cavernous sinus thrombosis, as well as epidural, subdural, and brain abscess. treatment of intracranial complications or impending intracranial complications requires surgical drainage of the involved sinus and concurrent treatment of the intracranial lesion by a neurosurgeon. 30 fungal sinusitis may occur in immunocompromised children, specifically severe diabetics, children undergoing chemotherapy, and bone marrow transplant recipients. the treatment of fungal sinusitis involves surgical drainage and intravenous antifungal agents. however, a chronic form of fungal sinusitis is allergic fungal sinusitis. these patients usually have other signs of allergy, such as asthma. the treatment of this condition is corticosteroids and dã©bridement of the involved sinuses. the diagnosis is made by sinus ct findings and the presence of eosinophils as well as fungi in the sinus secretions that are removed at the time of surgery. 11 congenital stenosis of the anterior bony aperture causes partial nasal obstruction that may be severe enough to cause difficulty feeding, respiratory distress, and failure to thrive. anterior rhinoscopy demonstrates a very constricted nasal opening bilaterally. ct of the nose shows marked narrowing of the pyriform aperture. neonates are obligate nasal breathers, and severe stenosis must be surgically corrected. because the stenotic segment is very anterior and the remainder of the nasal cavity is normal, removal of the constricting bone with drills is done through a sublabial approach. the nasal openings are stented with 3.0-mm endotracheal tube stents that are sutured in place and removed after a few days. choanal atresia may be unilateral or bilateral. the obstructing tissue is usually a bony plate, but a few cases will have only membranous atresia. unilateral choanal atresia presents as chronic unilateral rhinorrhea. there is no significant respiratory distress. because neonates are obligate nose breathers, bilateral choanal atresia is associated with severe respiratory distress, difficulty feeding, and failure to thrive. the diagnosis is suspected if catheters cannot be passed through the nose and into the pharynx. the obstruction may be visualized with a narrow flexible nasopharyngoscope after the nasal cavity has been suctioned of mucus and the nasal mucosa has been constricted with a nasal decongestant (e.g., oxymetazoline). the diagnosis is best made with ct of the nasal cavity. ct will demonstrate the atresia, define the tissue (bony or membranous), and show the configuration of the entire nasal cavity. choanal atresia may be successfully treated by removing the obstructing tissue transnasally. curets, bone punches, and drills may all be effective to remove the atresia plate. however, when the bony plate is very thick and there is an extremely narrow posterior nasal cavity, a transpalatal repair is more direct. a transpalatal repair provides better access for more effective removal of the bony plate and posterior septum ( fig. 52-3 ). stents fashioned from endotracheal tubes are placed and secured with sutures to the septum. they are removed in several weeks. the stents must be moistened with saline and suctioned several times daily to prevent mucus plugging and acute respiratory distress. transpalatal repair of choanal atresia has a lower incidence of restenosis. 27[pp 196-205] nasal dermoid cysts or sinuses present in the midline of the nasal dorsum ( fig. 52-4 ). they usually appear as a round bump or a pit with hair present in the pit ( fig. 52-5 ). they also may become infected. nasal dermoid sinuses may extend through the nasal bones into the nasofrontal area and have an intracranial component. both ct and mri may be necessary to demonstrate the extent of the dermoid. surgical removal is required to prevent infection and recurrence. this may be done between ages 3 and 5 years if prior infection has not occurred. dermoids confined to the nose are resected completely using a midline incision with an ellipse around the sinus tract. otolaryngologic disorders the tract is followed to its termination, and the nasal bones may need to be separated to reach the end of the tract. 27[pp 188-191] if an intracranial component is present, a combined craniotomy and nasal approach with a neurosurgeon is recommended. a nasal glioma presents as an intranasal mass and may be confused with a nasal polyp. the mass contains dysplastic brain tissue and may have an intracranial connection. ct and mri are important to define the extent of the glioma and intracranial component as well as to plan the surgical approach. an encephalocele presents as a soft compressible mass and may also be confused with a nasal polyp. intranasal encephaloceles extend through a defect in the skull at the cribriform plate. ct and mri define the extent of the encephalocele and are necessary to design the surgical approach. surgical removal often includes a frontal craniotomy. nasal encephaloceles may be associated with csf rhinorrhea and meningitis. an infant may be born with the soft nasal bones and the septum deviated to one side either as a result of a difficult delivery or from persistent intrauterine compression of the nose. the nasal structures can most often be returned to the midline with digital manipulation. if the nasal deformity is partially reduced, the nose usually straightens with growth during the first year to 18 months of age. nasal bone and nasal septal fractures in older children usually occur from a blow to the face during sports. there is usually a brief period of epistaxis and deviation of the nasal dorsum to one side. swelling occurs rapidly, and the degree of the cosmetic deformity or the need for fracture reduction may not be easily determined. at the fourth to sixth day after injury, the edema subsides and the need for reduction can be determined. nasal bone radiographs are of little help in making this judgment, so the need for nasal fracture reduction is usually based solely on clinical examination. effective nasal fracture reduction may be done up to 2 weeks after the injury. closed reduction under general anesthesia is the method of choice. oral antibiotics prevent infection and are essential if nasal packing is used to support the nasal bone. although nasal fracture reduction is not urgent, a septal hematoma from a fractured septum should be excluded by the initial physician seeing the child. a septal hematoma that remains untreated may cause cartilage necrosis and loss of nasal support, with a resulting saddle-nose deformity. treatment of a septal hematoma is with incision and evacuation of the clot. the mucoperichondral flap should then be sutured in place by bolster sutures through the septum. a small rubber band drain should remain in place for 12 to 24 hours, and antibiotics should be given. epistaxis in children usually occurs in little's area of the anterior septum and frequently results from digital trauma (nose picking). the bleeding usually stops with pressure by squeezing the nasal ala. infrequently, cauterization of the vessels under general anesthesia is needed. children may be observed inserting a foreign body into their nose, or they may inform their parents of the event. most children, however, present with a foul-smelling unilateral purulent nasal discharge and deny putting anything into their nose. most nasal foreign bodies are painless and do no harm to the nose except cause a foul nasal discharge. disc batteries, on the other hand, cause very rapid alkali burns of the nasal cavity and pain. batteries must be removed from the nose quickly because the chemical burn occurs in minutes to hours. if extensive tissue necrosis occurs, it may cause a nasal stenosis. removal of a nasal foreign body is aided by decongesting the nasal mucosa and using a headlamp to visualize the foreign body. a variety of forceps or hooks may be used. if the object is deep in the nose, the removal is best performed under general anesthesia. the endotracheal tube prevents aspiration of the object into the tracheobronchial tree if it is pushed back into the nasopharynx. one must remember that multiple foreign bodies may be present. nasal lacerations should be closed with care to match edges and restore the contours of the nose. standard wound closure technique is employed. the nasal mucosa does not need to be sutured unless a large flap is displaced. rhabdomyosarcoma, lymphoma, squamous cell carcinoma, and esthesioneuroblastoma may occur in the nose and sinuses of children. fortunately, these malignant tumors are very rare in children. the treatment of children with malignant tumors of the nose and sinuses usually involves a multidisciplinary, multimodal approach. juvenile nasopharyngeal angiofibroma is a benign tumor of adolescent males that originates from the lateral wall of the nose and nasopharynx. the tumor may completely obstruct the nose and fill the nasopharynx. this type of angiofibroma may also extend intracranially through the base of the skull. patients with these tumors present with nasal obstruction, recurrent epistaxis, and rhinorrhea. the tumor may be seen with a flexible fiberoptic nasopharyngoscope or a rod lens telescope after decongesting the nasal mucosa. it appears as a smooth reddish mass. biopsy of the mass should be avoided because of the potential for severe bleeding. ct and mri define the extent and location of the tumor. mr angiography helps to delineate the blood supply, which may originate from both the internal and external carotid arteries. contrast angiography may be reserved for presurgical planning and embolization of the copious blood supply that is often present. the treatment of juvenile nasopharyngeal angiofibroma is complete surgical resection after preoperative embolization. depending on the material used, the embolization may be effective for days to weeks. a variety of surgical approaches may be used, including endoscopic resection of small tumors. extensive tumors may require a combined midfacial and craniotomy approach. 29 some authors have proposed radiation therapy as the primary treatment of juvenile nasopharyngeal angiofibroma, but many surgeons are concerned about the long-term effects of radiation in children, including the induction of malignant tumors. the boundaries of the oral cavity include the lips anteriorly, the cheeks laterally, and the palate superiorly. the posterior boundary is a plane that extends from the soft palate to the junction of the anterior two thirds and posterior one third of the tongue. the oral cavity is composed of the vestibule, the space between the lips and cheeks and alveolar ridges, and the oral cavity proper. the vestibule and oral cavity proper are separated by the alveolar ridge and teeth. the vestibule is divided in the midline by the labial frenula of the upper and lower lips. the alveolar ridge is contiguous superiorly with the hard palate. the parotid ducts (stensen's ducts) enter the vestibule opposite the second maxillary molars. the submandibular ducts (wharton's ducts) enter the floor of mouth near the lingual frenulum. the palate is formed by a fusion of the primary palate anteriorly and medial growth of the palatal processes that form the secondary palate. the hard palate divides the nasal and oral cavities and is formed by the premaxilla and the horizontal plates of the palatine bones. the soft palate is formed by a muscular aponeurosis of the tensor veli palatini tendon. five muscles insert into this aponeurosis and include the tensor veli palatini, levator veli palatini, palatoglossus, palatopharyngeus, and the musculus uvulae. defects in formation of the hard and/or soft palate result in clefting. the sensory and motor innervation of the palate is through the trigeminal nerve and pharyngeal plexus. the circumvallate papillae divide the tongue into the anterior two thirds that lies in the oral cavity and the posterior one third lying in the oropharynx. the innervation and vascular supply to the two major divisions of the tongue reflect their differences in origin-the anterior two thirds of the tongue being a first branchial arch derivative (trigeminal) whereas the posterior one third being a combination of third and fourth arch derivatives (pharyngeal plexus). the hypoglossal nerve supplies motor innervation to the intrinsic musculature. in addition to the intrinsic tongue musculature, the action of four extrinsic muscles combine to provide mobility. the genioglossus protrudes and depresses, the hyoglossus retracts and depresses, the styloglossus retracts, and the palatoglossus elevates. in addition to the circumvallate papilla, other taste buds on the tongue surface include conical, filiform, fungiform, and foliate papillae. the pharynx is a fibromuscular tube that extends from the skull base to the level of the cricoid cartilage of the larynx and can be divided into three levels. the nasopharynx extends from the skull base to the level of the soft palate, the oropharynx extends from the soft palate to the tongue base, and the hypopharynx extends from the tongue base to the cricoid cartilage. three muscular constrictors combine to form the muscular portion of the pharynx: superior, middle, and inferior constrictors. passavant's ridge is a muscular segment of the superior constrictor that is involved in velopharyngeal closure. lower fibers of the inferior constrictor help to form the upper esophageal sphincter. the motor and sensory innervation of the pharynx is from the glossopharyngeal and vagus nerves via the pharyngeal plexus. a collection of lymphoid tissue within the pharynx forms waldeyer's ring that includes the palatine tonsils, the adenoid (pharyngeal tonsil), and lymphoid follicles lining the lateral and posterior pharyngeal walls. in addition to the acute onset of sore throat, viral pharyngitis typically presents with fever and malaise. signs include erythema of the pharynx and cervical lymphadenopathy. depending on the viral agent, associated symptoms of nasal obstruction and rhinorrhea may also be present. rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, adenovirus, and influenza virus are agents responsible for viral pharyngitis. primary herpetic gingivostomatitis, caused by herpes simplex types 1 or 2, presents as fever, adenopathy, and vesicles and ulcers on the lips, tongue, buccal mucosa, soft palate, and pharyngeal mucosa. herpangina and hand-foot-and-mouth disease are viral infections that involve the oropharynx. epstein-barr virus (ebv) infection (infectious mononucleosis) presents as acute pharyngotonsillitis, fever, generalized adenopathy, malaise, and splenomegaly. although ebv infection is suspected by the appearance of 10% or more atypical lymphocytes on a complete blood cell count and the presence of a positive monospot test, the definitive diagnosis is confirmed by elevated titers of ebv. group a î²-hemolytic streptococcus (gabhs, i.e., s. pyogenes) commonly infects the pharynx. in addition to sore throat, associated symptoms include fever, headache, and abdominal pain. associated signs include pharyngeal erythema, halitosis, tonsillar exudates, and tender adenopathy. diagnosis may be confirmed initially with a rapid streptococcal antigen test. because rapid antigen testing is more sensitive than formal plating on blood agar, a negative test does not need confirmation, but positive rapid streptococcal tests should be confirmed with formal plating. other bacterial pathogens that cause acute pharyngitis include haemophilus influenzae and groups c and g î²-hemolytic streptococci. occasionally, concurrent infection with penicillin-resistant staphylococcus aureus may interfere with treatment of a gabhs infection. 28 although many cases of gabhs infections respond to treatment with penicillin v or amoxicillin, emerging resistance to oropharyngeal pathogens mandates treatment of recalcitrant cases with an antibiotic having known effectiveness against î²-lactamase-producing organisms. in cases in which a lack of compliance is suspected, intramuscular benzathine penicillin or ceftriaxone may be used. acute pharyngitis may also be associated with acute bacterial infections of the nose, nasopharynx, and sinuses. these infections may be caused by a variety of viral and bacterial pathogens; and in addition to sore throat, symptoms include fever, mucopurulent nasal drainage, nasal obstruction, and facial pain. recurrent infection of the pharynx may be either viral or bacterial. gabhs are the most worrisome bacterial organisms because recurrent infection may lead to complications such as scarlet fever, acute rheumatic fever, septic arthritis, and acute glomerulonephritis. in addition to a history of multiple positive cultures for s. pyogenes, elevated antistreptolysin-o (aso) titers may identify patients with chronic infection who are at risk for developing complications. some asymptomatic children may be chronic carriers of gabhs, and elevated aso titers may not be a reliable indicator for distinguishing between an active infection and the carrier state. treatment of recurrent streptococcal infection or the child who is a carrier should include a trial course of an antibiotic shown to reduce carriage (e.g., clindamycin, vancomycin, or rifampin). children with recurrent pharyngotonsillitis unresponsive to medical therapy or those who suffer a complication should be considered for surgical management. whereas treatment of each child should be individualized, suggested guidelines for surgical candidates include seven infections in 1 year, five or more infections per year for 2 years, or three or more infections per year for 3 years. 24 other factors to be considered in employing a surgical option include severity of infection, response to antibiotic therapy, loss of time from school, and need for hospitalization. the pharynx and specifically the tonsils may be the target of chronic infection. affected children complain of chronic throat pain, halitosis, and production of white particles or tonsilliths. signs include erythema of the tonsils, cryptic debris, and chronically enlarged cervical adenopathy. a variety of viral and bacterial agents can be blamed for chronic infection of the pharynx. cultures may or may not be positive in these patients, because surface cultures may be negative while core tissue is positive. antibiotic therapy directed at anaerobes or s. aureus may be helpful in resistant cases. children with infections unresponsive to medical management are candidates for tonsillectomy. localized extension of tonsillar infection may result in peritonsillar cellulitis. the same pathogens that cause acute pharyngotonsillitis are responsible for peritonsillar cellulitis. in addition to severe sore throat, symptoms and signs include drooling, trismus, muffled voice, ipsilateral referred otalgia, and tender lymphadenopathy. the affected tonsil is usually displaced in a medial and inferior position. peritonsillar cellulitis may progress to frank abscess formation (quinsy). early cases of peritonsillar cellulitis may respond to oral antibiotics, such as the penicillins, cephalosporins, erythromycins, or clindamycin. unresponsive cases of cellulitis or abscess should be treated with intravenous antibiotics. in children with suspected abscess formation, a variety of surgical drainage procedures can be performed. needle aspiration or incision and drainage have been shown to be equally effective. 15 in persistent cases or in those children who will require general anesthesia for drainage, consideration should be given to performing a tonsillectomy (quinsy tonsillectomy). signs and symptoms of deep neck space (retropharyngeal/ parapharyngeal) infections that involve the pharynx typically present as fever, drooling, irritability, decreased oral intake, torticollis, and/or trismus. often there is a history of a preceding viral illness. stridor or symptoms of upper airway obstruction may be seen in half of patients. 32 a neck mass or enlarged cervical nodes may be present depending on the location of the infection. usual pathogens include coagulase-positive staphylococci and gabhs. anaerobic bacteria have been found in as many as 50% of cases. 32 complications of deep neck space infections include airway obstruction, bacteremia, rupture of the abscess into the pharynx with aspiration, mediastinal extension of infection, jugular thrombosis, and carotid artery rupture. in suspected cases, the diagnosis of a retropharyngeal/parapharyngeal space infection is confirmed with either contrast medium-enhanced ct or mri. widening of the retropharynx on a lateral neck radiograph suggests a retropharyngeal infection. while ultrasound can detect the presence of an abscess cavity, ct or mri are most helpful in demonstrating the extent of infection and the location of surrounding structures of importance, specifically the great vessels. contrast medium-enhanced ct is particularly useful in distinguishing a phlegmon (cellulitis) from cases of frank suppuration. demonstration of a hypodense region with surrounding rim enhancement has been shown to correlate with an abscess in 92% of cases (fig. 52-6) . the initial management of a deep neck infection should begin with intravenous antibiotics, including oxacillin, clindamycin, cefazolin, î²-lactamase penicillins, or a combination thereof. surgical drainage should be reserved for those children who fail to show clinical improvement or progress to frank abscess formation on ct. the usual approach to surgical drainage is intraoral if the abscess points medial to the great vessels or extraoral if the infection points lateral to the great vessels. complications of deep neck infections should be treated aggressively. mediastinal spread requires prompt surgical drainage in most cases. an infected jugular thrombosis (lemierre's syndrome) can be a source of metastatic spread of infection as septic emboli. signs and symptoms include spiking chills and fever (picket-fence fevers) and a neck mass in spite of appropriate antibiotic therapy. ligature or excision of the infected thrombus may be required to eradicate the infection. in the past decade, the impact of sleep-disordered breathing (sdb) on the health of children has been well described, beginning with the report of normative sleep data by marcus and colleagues. 20 children appear to have briefer but more frequent episodes of partial (hypopnea) and complete (apnea) obstruction. because an apnea of less than 10 seconds may represent several missed breaths in a child, an apnea of any duration is abnormal. in most cases the site of obstruction during sleep is in the pharynx. in contrast to adults with this disorder in whom the pharyngeal impingement is due to adipose tissue surrounding the pharyngeal musculature, the major cause of airway obstruction in children results from adenotonsillar hypertrophy. the apnea index represents the number of apneas in an hour, with a normal value being less than 1 in children. because most children have an increased frequency of partial obstructions compared with adults, a measure of hypopneas may be more significant. a hypopnea is variably described as a reduction in airflow or respiratory otolaryngologic disorders effort or oxygen desaturation or combination thereof. respiratory disturbance index is a measure of both apneas and hypopneas in an hour and may be a better reflection of sdb in children. a respiratory disturbance index greater than 5 is abnormal. upper airway resistance syndrome represents obstructed breathing with normal respiratory indices but with sleep fragmentation and electroencephalographic arousals that indicate disordered sleep. the major group at risk for sdb includes children with adenotonsillar hypertrophy secondary to lymphoid hyperplasia (figs. 52-7 and 52-8). whereas the age of affected children ranges from 2 years through adolescence, the prevalence mirrors the age of greatest lymphoid hyperplasia, 2 to 6 years, the age the tonsils and adenoids are largest in size. other at-risk groups include syndromic children with down syndrome, children with craniofacial disorders, and patients with cleft palate or storage diseases (hunter's, hurler's syndromes). adverse effects of obstructive sleep apnea on children include poor school performance, failure to thrive, facial and dental maldevelopment, and, rarely, severe cardiac impairment, including systemic hypertension, cardiac arrhythmias, and cor pulmonale. daytime symptoms include noisy mouth-breathing, nasal obstruction and congestion, hyponasal speech, and dyspnea on exertion. in contrast to adults, hypersomnolence is uncommon in children because of the lower incidence of gas exchange abnormalities, specifically hypercarbia. children may complain of headaches, seem irritable, and perform poorly in school. nighttime symptoms are more obvious and include snoring, gasping and choking respirations, apnea, coughing, and a variety of other behaviors including sleepwalking, sleeptalking, rocking, head banging, and bruxism. enuresis may appear in children with airway obstruction and then resolve after surgical treatment. in addition to enlarged tonsils, signs include the presence of a posterior pharyngeal flap in cleft palate patients, a craniofacial disorder, adenoid facies, and, rarely, evidence of right-sided heart failure. the diagnosis of sdb is suggested by history and physical examination. confirmation of obstruction and apnea may be made with overnight pulse oximetry and video or audio monitoring of sleep. the "gold standard" in the diagnosis of obstructive sleep apnea remains formal polysomnography, including measures of nasal and oral airflow, chest wall movements, electrocardiography, extraocular muscle movements, and gastric ph monitoring in selected cases. depending on the suspected site of obstruction, adjuvant studies such as a lateral neck radiograph, mri of the head and neck, and flexible upper airway endoscopy might be helpful. the nonsurgical management of sdb consists of weight loss in obese patients and treatment of underlying allergies and gastroesophageal reflux. nasal and dental appliances to maintain airway patency that may be useful in adults are usually poorly tolerated in children. nasal continuous positive airway pressure, the mainstay of treatment in adults, is tolerated in many children and should be considered as a treatment option, especially in patients in whom other therapies have been exhausted or proven ineffective. the initial surgical treatment for most children with sdb remains a tonsillectomy and adenoidectomy, a therapy that is usually curative. in patients with documented sleep apnea or a sleep disorder, both procedures should be utilized even if the tonsils appear small. tonsillectomy and adenoidectomy techniques that have been standard for decades have been supplanted in some institutions by new technology including use of coblation, harmonic scalpel, and the microdebrider. efficacy of these newer techniques over established methods remains unproven. complications after tonsillectomy and adenoidectomy usually consist of respiratory compromise and acute or delayed bleeding. since the advent of modern pediatric anesthesia, respiratory complications such as aspiration with resultant pneumonia and lung abscess are rare. humidification, corticosteroids, and antibiotics have all been shown to improve the postoperative course after tonsil and adenoid surgery. young children are most vulnerable to complications, and in most institutions children younger than 4 years of age are observed overnight for signs of dehydration and respiratory compromise. adjuvant surgery in the management of sdb includes craniofacial repair or posterior flap revision surgery in appropriate patients. midface, mandibular, and hyoid advancement have proved useful in selected patients, along with nasal surgery such as septoplasty, partial inferior turbinectomy, or nasal polypectomy. tracheostomy remains the treatment of last resort in patients who fail to respond to other forms of therapy. ankyloglossia or tongue-tie is a common congenital disorder involving the lingual frenulum ( fig. 52-9 ). neonates with diminished tongue mobility due to a foreshortened frenulum may have problems in sucking and feeding. because the frenulum is thin and relatively avascular in neonates and young infants, it can often be incised as an office procedure. in older children the greatest effect of ankyloglossia is on speech. because the tip of the tongue curls under on extrusion and has limited lateral and superior movement, speech articulation may be affected. surgical treatment in these patients may require a short general anesthetic as the frenulum is thicker and more vascular, requiring surgical correction that includes either simple division with or without a z-plasty repair. macroglossia is uncommon. generalized macroglossia, as seen in association with omphalocele, visceromegaly, and adrenal and renal disorders (beckwith-wiedemann syndrome), with glycogen storage diseases (hunter's and hurler's syndromes) or hypothyroidism, is rare. relative macroglossia can be seen normally on occasion but is most common in down syndrome. the most serious complication of this condition is airway obstruction. in infants, macroglossia should be distinguished from focal enlargement of the tongue seen in patients with a lymphatic malformation or hemangioma. glossoptosis, posterior displacement of a normal-sized tongue, is seen in association with cleft palate and micrognathia in infants afflicted with the pierre robin sequence. infants with airway obstruction secondary to an enlarged or displaced tongue may require a tracheostomy. macroglossia in older children that affects cosmesis, interferes with speech, or causes drooling may be treated with a variety of tongue reduction techniques. epulis is a congenital granular cell tumor that typically presents as a soft, pink submucosal mass on the anterior alveolar ridge of the maxilla (fig. 52-10) . females are otolaryngologic disorders more commonly affected, and symptoms are usually confined to feeding problems. surgical excision is curative. ranula is a pseudocyst located in the floor of the mouth that may occur congenitally or result from intraoral trauma (fig. 52-11) . large ranulas may extend through the mylohyoid musculature and present in the neck as a "plunging ranula." treatment of ranulas is by excision or marsupialization of the pseudocyst, often in conjunction with excision of the sublingual gland. mucoceles are also pseudocysts of minor salivary gland origin and frequently rupture spontaneously. recurrent or symptomatic mucoceles respond to surgical excision. hemangioma is a proliferative endothelial lesion found commonly in the head and neck. their growth characteristics include enlargement during the first year of life, followed by spontaneous resolution. surgical excision or treatment with corticosteroids may be necessary in lesions that cause ulceration and bleeding, airway obstruction, cardiovascular compromise, or platelettrapping coagulopathy (kasabach-merritt syndrome). vascular malformations, including venous, arterial, or arteriovenous malformations, rarely occur in the oral cavity and pharynx and necessitate intervention only if they cause pain, bleeding, ulceration, or heart failure. management of complicated cases is by surgical excision or sclerotherapy for low-flow lesions (venous) and angiographic embolization for high-flow lesions. lymphatic malformation, formerly known as lymphangioma or cystic hygroma, is congenital and usually presents before 2 years of age. histologically, lymphatic malformations consist of multiple dilated lymphatic channels or may contain either capillary or venous elements (venolymphatic malformations). lymphatic malformations can occur anywhere in the neck and may cause extensive cosmetic deformity and functional problems in cases with involvement of the tongue, floor of mouth, mandible, or larynx. surgical resection of lymphatic malformations may be fraught with difficulty because they lack a capsule and are infiltrative. during surgical excision, care should be taken to avoid damaging nearby vital structures, and debulking is an acceptable option to total radical excision in many cases. postoperative suction drains can be helpful in preventing the recurrence of lymphatic drainage under skin flaps. carbon dioxide laser therapy has been employed in superficial lymphatic malformations of the tongue, and sclerotherapy of large cystic lesions may be an option. foregut cysts are true cysts, lined with respiratory epithelium, that present in the floor of mouth and should be distinguished from dermoid cysts, lined with stratified squamous epithelium and skin appendages, which may also be found in this location. a thyroglossal duct cyst may rarely present in the base of the tongue. likewise, aberrant thyroid tissue, lingual thyroid, presents as a purple mass in the tongue base. thyroid tissue in this location is usually hypofunctioning, and affected children require thyroid supplementation. other aberrant rests of tissue, choristomas, consist of gastric, enteric, or neural tissue of normal histology in an abnormal location. second branchial cleft derivatives will rarely present as a cystic mass near the superior pole of the tonsil. their extent and associated tracts can be demonstrated on mri. tornwaldt's cyst is a blind pouch in the nasopharynx that represents a persistence of an embryonic connection between the primitive notochord and the pharynx. other benign nasopharyngeal masses include nasopharyngeal teratomas, dermoid lesions (hairy polyp), and nasopharyngeal encephaloceles. most of these lesions are best evaluated by ct and/or mri to determine their extent and the presence of an intracranial connection. surgical excision is curative in most cases. squamous papillomas are benign slow-growing lesions typically found on the soft palate, uvula, and tonsillar pillars and are the result of infection with serotypes 6, 14, or 22 of the human papillomavirus (hpv). because of concern that these lesions could spread to the larynx or trachea, complete surgical excision is usually recommended. pleomorphic adenoma (mixed tumor) is a benign neoplasm of minor salivary glands with a predilection for the palate, although it may also be found in the lip and buccal mucosa. treatment is with surgical excision. rhabdomyosarcoma, the most frequent soft tissue malignancy of childhood, typically occurs in the 2-to 6-year age group and is derived from embryonic skeletal muscle. 4, 18 in the oral cavity and oropharynx it presents as a rapidly growing mass in the tongue, palate, and uvula or cheek. these tumors metastasize early to local nodes, lung, and bone. surgical therapy is limited to biopsy, excision of small lesions, or surgical salvage of treatment failures. the usual therapy includes a combination of chemotherapy and radiation therapy. lymphoma of the oral cavity and oropharynx typically involves the lymphoid tissue of waldeyer's ring and presents as a mass of the tonsil or in the nasopharynx. 8 the diagnosis may be suspected by evidence of involved adenopathy in the neck but is confirmed by surgical biopsy. treatment is with a combination of chemotherapy and radiation therapy. other rare malignant neoplasms of the oral cavity and pharynx include malignant salivary gland tumors (mucoepidermoid carcinoma) and epidermoid or squamous cell carcinoma. this latter tumor has been reported in organ transplant patients and adolescents who use snuff or chewing tobacco. 19 treatment is usually surgical depending on the site and extent of involvement. with the exception of the hyoid bone, the major structural framework of the larynx consists of cartilage and soft tissue. the hyoid bone lies superior to the larynx and is attached to it by the thyrohyoid membrane and strap muscles. the hyoid bone is derived from the second and third branchial arches. the cartilaginous structures of the larynx are composed of hyaline cartilage, with the exception of the epiglottis, which is composed of elastic cartilage. the cartilaginous structures of the larynx develop from the fourth, fifth, and sixth branchial arches. there are nine laryngeal cartilages, three that are single (thyroid, cricoid, and epiglottis) and six that are paired (arytenoid, cuneiform, and corniculate). the thyroid cartilage consists of two quadrilateral cartilages that form the anterior framework of the larynx. the cricoid cartilage is the only complete cartilaginous structure in the airway and provides posterior stability and a base of support for the cricoarytenoid and cricothyroid joints. the cricothyroid muscles are paired extrinsic laryngeal muscles that serve to tilt the larynx down and forward, tensing the vocal folds. paired intrinsic muscles-the thyroarytenoid, thyroepiglottic, and aryepiglottic muscles-act as a sphincter to close the larynx. the vocalis muscle comprises the internal fibers of the thyroarytenoid muscle and attaches to the vocal ligament. action of this muscle serves to regulate the pitch of the vocal ligament. the other set of paired muscles includes the posterior cricoarytenoid, lateral cricoarytenoid, and interarytenoid muscles. the posterior cricoarytenoid muscles serve to abduct the vocal folds, whereas the cricoarytenoid and interarytenoid muscles adduct the vocal folds. the quadrangular membrane is a connective tissue covering of the superior larynx that ends in a free margin along the vestibular ligament of the false cord. the conus elasticus is a membrane of elastic tissue that extends superiorly from the cricoid cartilage to form the paired vocal ligaments, the supporting structures of the vocal folds. the blood supply of the larynx arises from the superior and inferior laryngeal arteries. the former is a branch of the superior thyroid artery, whereas the latter is a branch from the thyrocervical trunk. the intrinsic muscles of the larynx are innervated by the recurrent laryngeal nerve, which also supplies sensory branches to the inferior larynx. the superior laryngeal nerve has two branches: the external branch innervates the cricothyroid muscle, while the internal branch supplies sensation to the superior larynx. the larynx has multiple functions within the upper airway. during respiration, it regulates airflow by opening during inspiration. the posterior cricoarytenoid muscle contracts with each inspiration to abduct the cords just before activation of the diaphragm. the protective function of the larynx produces two reflexes: cough and closure. cough is important to expel mucus and foreign objects. the closure reflex serves to prevent aspiration of foreign matter. in addition to closure, the larynx elevates during swallowing. both closure and elevation occur simultaneously along with relaxation of the cricopharyngeus muscle during the swallow of a bolus. finally, the larynx plays an important role in speech production by generating sound. vibration of the mucosa covering the vocalis structures produces sound whose pitch and register is altered by changes in tension, length, and mass of the underlying vocalis muscle and ligament. the larynx of an infant sits much higher than that of an adult. the cricoid is located at the level of c4, whereas the tip of the epiglottis is at c1. the close approximation of the epiglottis to the soft palate makes the infant an obligate nose breather. by 2 years of age, the larynx has descended to the level of c5 and reaches the adult level of c6 to c7 by puberty. the glottis of the newborn is 7 mm in the anteroposterior dimension and 4 mm in the lateral dimension. the narrowest area of the infant airway, the subglottis, is approximately 4 mm in diameter. symptoms of acute airway obstruction include dyspnea, cough, vocal changes, dysphagia, and sore throat. dyspnea and rapid or labored breathing are indications of inadequate ventilation and may be triggered by changes in pco 2 and po 2 . a stimulus anywhere in the airway may produce cough. it is difficult to localize the site of the stimulus from the quality of the cough. changes in the child's vocal character such as hoarseness or a muffled or weak cry may help in localizing the area of obstruction. dysphagia for solids and/or liquids is often associated with airway obstruction. depending on the cause of airway obstruction, affected patients may complain of sore throat. the child's overall appearance is the first sign to be assessed in airway obstruction, because airway status often dictates how quickly further evaluation and intervention need to be performed. the level of consciousness should be determined because the unconscious or obtunded patient may need immediate airway management. along with cyanosis in a patient without cyanotic heart disease, the presence of anxiety, restlessness, and diaphoresis are all ominous signs of impending airway compromise. other symptoms of airway obstruction include tachypnea and substernal retractions. the child with airway obstruction is often tachycardic. the presence of bradycardia is a otolaryngologic disorders late indicator of severe hypoxia. the presence of a muffled cry often suggests obstruction at the level of the pharynx, whereas a barking cough is associated with laryngeal inflammation and edema. stertor is a snorting sound whose origin is often in the pharynx. stridor is noise produced by turbulent airflow in the laryngeal or tracheal airway. inspiratory stridor suggests turbulence at or above the glottis. expiratory stridor results from turbulent airflow in the distal trachea or bronchi. biphasic stridor suggests a tracheal source. the degree and loudness of the sound is not always indicative of the severity of obstruction, because stridor can become softer just before complete obstruction. other important signs of airway obstruction include drooling and use of accessory respiratory muscles. in addition to determination of the child's physical status, assessment of the degree of airway obstruction should include an evaluation of the ventilatory status. pulse oximetry provides an immediate record of arterial oxygenation while transcutaneous monitoring of co 2 is a good indicator of ventilation. the lateral neck radiograph remains the best study for the initial evaluation of a child with airway obstruction because it demonstrates the anatomy from the tip of the nose to the thoracic inlet. the anteroposterior view of the neck is also helpful, specifically in defining areas of narrowing, such as a steeple sign associated with subglottic edema. a chest radiograph is also important in the initial assessment to identify foreign bodies or other conditions such as unilateral emphysema, atelectasis, or pneumonia that may account for the child's respiratory compromise. if time permits, a barium swallow or airway fluoroscopy may provide additional information. additional airway evaluation may include a brief flexible endoscopic examination. the nose is first sprayed with a combination of 2% lidocaine and oxymetazoline, and the child is gently restrained. the airway can be examined from the nares to the glottis. attempts to pass a flexible scope through the glottis in a child with airway obstruction should be avoided. likewise, flexible endoscopy should be avoided in a child with supraglottitis because of the possibility of precipitating complete obstruction. children with suspected airway pathology distal to the glottis or those in whom the possibility that flexible endoscopy could compromise the airway should undergo any airway examination in the operating room where rigid endoscopes and other airway equipment is immediately available to secure the airway if necessary. nonsurgical intervention in the child with acute airway obstruction may begin with just observation alone in a high surveillance unit. humidified oxygen administered by face mask will improve po 2 and clearance of secretions. racemic epinephrine administered by nebulizer acts to reduce mucosal edema and is useful in conditions such as laryngotracheobronchitis (infectious croup). because its length of action lasts 30 to 60 minutes, treated patients should be observed for signs of rebound for 4 to 6 hours after administration. corticosteroids have been shown to have value in the management of postintubation croup, adenotonsillar hypertrophy that results from ebv infection, allergic edema, and spasmodic croup. use of corticosteroids in infectious croup and in infants with a subglottic hemangioma remains controversial. 10, 16 other adjuvant therapies include antibiotics and inhalation of helium/oxygen mixture (heliox). although viral agents are often responsible for inflammation in the larynx and trachea, bacterial superinfection is also common. because of the prevalence of penicillin-resistant organisms, broad-spectrum antibiotics, including a higher-generation cephalosporin, penicillinase-resistant penicillin, or î²-lactamase penicillin, are useful in preventing or eradicating infection. heliox is a mixture of gas in which helium is used to replace nitrogen. the advantage of the helium-oxygen mixture is that its low density reduces air turbulence and gas resistance, allowing improved delivery of oxygen in patients with airway obstruction. nonsurgical airway management may include use of nasal or oral airways, endotracheal intubation, and, rarely, transtracheal ventilation. nasal airways of rubber or other synthetic material can be easily inserted into the nose of most children after adequate lubrication with a water-soluble lubricant. their best use is in cases where the pharynx is the site of obstruction. oral airways are not as readily tolerated by children and only serve as a brief solution to an airway problem. during the 1970s, endotracheal intubation with polyvinyl chloride tubes revolutionized the management of supraglottitis, and even today intubation remains the mainstay of initial airway therapy in most children with severe airway obstruction. the size of the endotracheal tube used correlates with the age of the child. the subglottis, the narrowest part of the infant airway, typically admits a 3.5-or 4.0-mm inner diameter tube. the tube used in children older than a year can be roughly estimated by using the following formula: tube size = (16 + age in years) ã· 4. once the airway has been established, the tube should be carefully secured and the child appropriately sedated and/or restrained if necessary to avoid accidental self-extubation. another method of airway management should be considered in children with an unstable cervical spine or in whom oral or neck trauma makes visualization difficult. transtracheal ventilation, insertion of a 16-gauge needle through the cricothyroid membrane for the delivery of oxygen, should be reserved for emergencies and used only until a more stable airway can be obtained. the surgical management of the child with acute airway obstruction should begin with endoscopy. the larynx can be visualized with one of a variety of pediatric laryngoscopes and the airway secured with a rigid pediatric ventilating bronchoscope of appropriate size. once the airway is secured, a more stable form of airway management can be utilized. rarely, in a child with acute airway obstruction, an airway cannot be established, and a cricothyrotomy may need to be performed. as in adults, this procedure avoids some of the risks of bleeding and pneumothorax inherent in a formal emergency tracheostomy. a small endotracheal or tracheostomy tube can be inserted through the incision in the cricothyroid membrane, but conversion should be made to a more stable airway as soon as possible. tracheostomy remains the preferred airway in cases of acute obstruction in which a translaryngeal approach is unsuccessful or must be avoided. the emergent tracheostomy should be avoided if at all possible to lessen complications of bleeding, pneumothorax, pneumomediastinum, subcutaneous emphysema, or damage to surrounding structures. the incidence of these complications can be reduced by careful attention to surgical technique, good lighting, and adequate assistance. laryngomalacia is the most common cause of newborn stridor and is caused by prolapse of the supraglottic structures (arytenoid cartilages, aryepiglottic folds) during inspiration ( fig. 52-12 ). symptoms typically appear at birth or soon thereafter and include inspiratory stridor, feeding difficulties, and, rarely, apnea or signs of severe airway obstruction. gastroesophageal reflux disease tends to worsen symptoms of laryngomalacia. the diagnosis is confirmed by flexible endoscopy of the larynx, and other airway pathology can be excluded with lateral neck, chest, and barium swallow radiography. in most cases, laryngomalacia is self-limited and resolves by 18 months of age. changes in positioning and feeding, treatment of reflux, and, in some neonates, use of monitoring may be necessary. in severe cases, surgical intervention with either a supraglottoplasy (surgical division of the aryepiglottic folds) or a tracheostomy may be necessary. tracheobronchomalacia is defined as collapse of the tracheobronchial airway. it may be congenital or acquired (from long-standing intubation and infection) and may be segmental or involve the entire tracheobronchial tree. depending on the extent and location, symptoms include low-pitched biphasic stridor and signs of respiratory compromise. the diagnosis is usually made with endoscopy, although fluoroscopy of the airway may often demonstrate it. treatment ranges from observation in most cases to airway management with a tracheostomy tube and positive-pressure ventilation in severe cases. vocal fold paralysis is the second most common congenital laryngeal anomaly (after laryngomalacia) and may be unilateral or bilateral. congenital vocal fold paralysis may be caused by neurologic abnormalities (hydrocephalus, arnold-chiari malformation), birth trauma, or rarely in association with neoplasms of the larynx or neck. neonates with bilateral involvement typically present with highpitched inspiratory or biphasic stridor but a good cry. respiratory compromise and feeding difficulties may accompany the stridor. in infants with unilateral involvement, the airway may be adequate although a few infants will show evidence of compromise, especially during feeding. the cry is often hoarse or breathy. acquired vocal fold paralysis may result from trauma or from neoplasms of the chest or neck or may be iatrogenic, typically after surgery of the neck or arch of the aorta. the diagnosis of unilateral or bilateral vocal fold paralysis is confirmed with endoscopy. additional studies in the evaluation of patients with vocal fold paralysis include lateral neck and chest radiography, barium swallow, and ct or mri of the head and neck. most cases of unilateral involvement can be observed, but infants with bilateral vocal fold paralysis often require a tracheostomy. in addition, infants with associated feeding difficulties may necessitate a gastrostomy. in older children (> 4 or 5 years of age) a more permanent solution such as a cordotomy or arytenoidectomy can be considered to improve the glottic airway. congenital subglottic stenosis is the third most common congenital laryngeal anomaly and is defined as a neonatal larynx that fails to admit a 3.5-mm endotracheal tube without a history of prior instrumentation or intubation ( fig. 52-13 ). the underlying abnormality is a cricoid cartilage that is either small or deformed. infants with congenital subglottic stenosis present with inspiratory or biphasic stridor, barking cough, and other symptoms of airway obstruction. the diagnosis is often suggested by narrowing of the subglottis on a lateral neck radiograph and confirmed by endoscopy. treatment depends on the severity of symptoms and ranges from observation to laryngeal reconstruction to tracheostomy. a child with a subglottic hemangioma presents with the onset of progressive stridor during the first few months of life (fig 52-14) . hemangiomas are proliferative endothelial lesions that can form in the submucosa of the posterior subglottis. occasionally, they may involve the subglottis in a circumferential pattern. associated cutaneous hemangiomas may be found in approximately 50% of patients. symptoms are dependent on the amount of airway compromise and include biphasic stridor, barking cough, difficulty feeding, and other symptoms and signs of airway obstruction. the diagnosis may be suggested on a lateral neck radiograph but is confirmed with endoscopy. nonsurgical management of infants with a subglottic hemangioma includes observation or treatment with systemic corticosteroids. surgical therapy includes laser excision, open excision through a laryngofissure, or a tracheostomy. a laryngocele is an air-filled dilatation of the saccule of the larynx that communicates with the laryngeal airway. it may present internally into the posterior superior false cord region or externally through the thyrohyoid membrane. a saccular cyst is fluid filled and protrudes between the true and false vocal folds. the diagnosis of this lesion is confirmed endoscopically, and ct of the larynx is helpful in assessing its extent and if it is fluid or air filled. treatment is with endoscopic marsupialization or excision through a laryngofissure. laryngotracheobronchitis (viral croup) is an inflammation of the subglottic airway caused by a variety of parainfluenza and influenza viral agents. the infection may involve the entire glottis and extend into the trachea and bronchi. affected children fall typically into the 1-to 3-year age group; males are more commonly affected than females. symptoms and signs of viral croup include biphasic stridor, barking cough, and hoarseness, often in association with a prodromal viral upper respiratory tract infection. the diagnosis of croup is made clinically, but endoscopic examination may help to exclude other pathologic processes. care should be taken not to instrument the subglottis, causing more swelling and inflammation and precipitating acute obstruction. lateral neck radiography demonstrates subglottic narrowing, whereas anteroposterior neck films show a "steeple sign," the result of subglottic edema. treatment of viral croup is typically supportive with humidification. use of corticosteroids remains controversial. treatment with nebulized racemic epinephrine in the emergency department or hospital setting often relieves symptoms; however, rebound of signs may occur several hours later and children should be monitored accordingly. severely affected children may require intubation for respiratory failure. a smaller than normal tube should be employed. in rare cases, a tracheostomy may be required if the inflammation fails to resolve. a child younger than 1 year of age with recurrent bouts of "croup" should be suspected of having either congenital subglottic stenosis or a hemangioma. spasmodic croup is the recurrence of croup-like symptoms in a child who is otherwise well. fever is rarely present, and the attacks frequently occur at night. gastroesophageal reflux disease has been suggested as a possible inciting process. treatment of spasmodic croup is usually observant, although corticosteroids or reflux medications may prove beneficial. supraglottitis (epiglottitis) is an infectious disease that involves the supraglottic larynx. in children the typical pathogen is type b haemophilus influenzae (hib). other pathogens have been implicated in adolescent and adult cases. the incidence of supraglottitis in children has diminished markedly since the introduction of the conjugated hib vaccine in the early 1990s. 17 affected children are somewhat older than those seen with croup-in the 3-to 6-year age group. symptoms and signs have a rapid onset, progress quickly to frank airway obstruction, and include stridor, dysphagia, fever, muffled voice, and signs of systemic toxicity. affected children frequently sit and assume the "sniffing" position in an attempt to maximize their airway. intraoral or endoscopic examination should be avoided in suspected patients because of concern for precipitating complete obstruction. lateral neck radiography demonstrates a classic "thumbprinting" of the epiglottis but should only be obtained if facilities are present in close proximity to secure the airway. prompt airway management is essential in children with supraglottitis. the child's airway should be secured in either the emergency department or operating room with team members who include a pediatrician, anesthesiologist, critical care physician, otolaryngologist, or pediatric surgeon or others familiar with the pediatric airway. after inducing the child with general anesthesia, the airway should be intubated. examination of the supraglottis may be made, and cultures of the larynx and blood are obtained. equipment to perform a tracheostomy should be readily available. the child should remain intubated for 24 to 72 hours and should be supported with intravenous fluids and antibiotics that treat antibioticresistant haemophilus (third-generation cephalosporins, chloramphenicol). bacterial tracheitis (membranous croup) often occurs as a complication of another infection, such as measles, varicella, or other viral agents. the most common organisms include s. aureus, gabhs, m. catarrhalis, or h. influenzae. it can occur in any age child and present with stridor, barking cough, and low-grade fever. symptoms and signs then progress to include high fever and increasing obstruction and toxicity. the diagnosis may be suspected by diffuse narrowing of the tracheal air shadow on chest radiograph but is confirmed by endoscopic examination in the operating room. purulent debris and crusts can be removed at this time. cultures of secretions and crusts may be helpful in guiding intravenous antibiotic therapy that should be aimed initially at the usual pathogens. the airway should be secured with an endotracheal tube or, rarely, a tracheostomy. repeat endoscopic examination of the airway may be warranted to continue dã©bridement and to determine the feasibility of extubation. the chronic management of subglottic stenosis and other prolonged airway disorders is discussed in chapter 63. recurrent respiratory papillomatosis is the most common benign neoplasm of the larynx in children. squamous papillomas involve the larynx and occasionally the trachea and lower respiratory tract as exophytic lesions. because of its recurrent nature, recurrent respiratory papillomatosis causes morbidity and, rarely, mortality due to malignant degeneration. patients may be almost any age, but the disease is more aggressive in children. human papillomavirus subtypes 6, 11, 16, and 18 have all been identified within papilloma tissue. the first two subtypes have been associated with genital warts, whereas the latter two have been associated with cervical and laryngeal cancers. the exact mechanism of human papillomavirus infection in the larynx remains unknown. transmission of virus to the child from a mother with genital warts is suspected in many cases, but there is no concrete evidence to support this route of infection. children afflicted with recurrent respiratory papillomatosis present initially with hoarseness but may also have symptoms and signs of airway obstruction, including stridor. lateral neck radiography may suggest laryngeal involvement, but the diagnosis is confirmed by direct laryngoscopy and biopsy (fig. 52-15 ). in addition to the trachea and bronchi, squamous papillomas may also be found in the oral cavity. surgical excision is the mainstay of therapy in patients with recurrent respiratory papillomatosis. in the past, papillomas were excised using the carbon dioxide laser. more recently, the laryngeal microdebrider has become the preferred method of excision in many centers. in aggressive cases with swift recurrence and accompanying airway obstruction, tracheostomy may be necessary for airway management, although tracheostomy has been implicated in the spread of disease to the trachea and lower respiratory tract. medical adjuvant therapy that has been employed with mixed results includes interferon, photodynamic therapy with dihematoporphyrin ether, indole-3-carbinol, or antiviral agents such as cidofovir. other benign laryngeal neoplasms are rare and include connective tissue tumors such as chondromas or fibromas, neurogenic tumors such as neurofibromas, or granular cell tumors and other cell types such as hamartomas or fibrous histiocytomas. malignant tumors of the larynx are also rare and include squamous cell carcinoma and a variety of epithelial and connective tissue malignancies such as spindle cell carcinoma, rhabdomyosarcoma, mucoepidermoid carcinoma, and chondrosarcoma. metastatic tumors and lymphoma may also rarely involve the larynx in children. diagnosis is suspected by the sudden appearance of stridor, hoarseness, and airway obstruction and confirmed by biopsy. treatment is dependent on cell type and may include surgical excision, radiation therapy, and/or chemotherapy. the surgical anatomy and embryology of the neck is discussed in chapter 56. the initial examination of a disease or disorder of the neck begins with a thorough history. a detailed history can often serve to focus the differential diagnosis of a neck disorder. the age of the child is an important otolaryngologic disorders first consideration. the appearance of a neck mass in an infant often suggests a congenital disorder, whereas the sudden appearance of a mass in an adolescent might suggest a malignant process. inflammatory diseases of the neck may occur in any age group but typically mirror the incidence of upper respiratory tract infections in children. growth and temporal relationships are often important clues to a diagnosis. neck masses that grow rapidly suggest either an inflammatory or malignant process, whereas slow-growing masses are typically benign. a history of systemic infection elsewhere in the body or recent travel or exposure to farm animals often points to an infectious origin. a history of trauma to the neck may explain the sudden appearance of a neck mass. likewise, changes in the size of a neck mass with eating may suggest a salivary gland origin. vascular lesions enlarge with straining or crying. finally, systemic symptoms of fever, weight loss, night sweats, or fatigue in association with the sudden development of a neck mass may indicate a malignant process. the physical examination of a child with a neck mass should begin with a comprehensive examination of the entire head and neck. because the vascular, neural, and lymphatic patterns of the head drain into the neck, the source of neck disorders may be found in the head. depending on the differential diagnosis, a physical examination of the entire body, including an assessment of lymph nodes in the groin and axillae and the presence of an enlarged spleen or liver, is essential. palpable lymph nodes in the neck of children are a common finding, but lymph nodes larger than 2 cm fall outside the range of normal hyperplastic nodes and should be either monitored or investigated. the sudden appearance of large nodes in either the posterior cervical or supraclavicular regions may suggest a malignancy. 31 the consistency of a neck mass is also important in narrowing the differential diagnosis. hard masses tend to be associated with either infection or malignancy. fixation of a neck mass to skin or nearby structures is also suggestive of a malignancy. cysts or abscesses tend to have a characteristic feel on palpation. depending on the differential diagnosis after a history and physical examination, radiologic studies may be useful. a lateral neck radiograph may demonstrate an abnormality of the nasopharynx, retropharynx, or cervical spine. likewise, a chest radiograph may identify a malignancy, sarcoidosis, or tuberculosis. infection or a neoplastic process in the sinuses may appear on a sinus series. ct and mri are useful in the evaluation of a neck mass. demonstration of hypodensity on ct suggests an inflammatory or necrotic process. ring enhancement of a hypodense region on a contrast ct scan is indicative of an abscess. mri is excellent for distinguishing fine detail within soft tissue and in the evaluation of vascular lesions of the neck. finally, ultrasound is helpful in distinguishing solid and cystic masses. use of ultrasound preoperatively in patients with a thyroglossal duct cyst is also a simple and economic way to assess the presence of normal thyroid tissue when it is not easily felt. ultrasound and thyroid scanning should be employed in the assessment of any thyroid mass. selected laboratory studies may be helpful in the evaluation of a child with a neck disorder. a complete blood cell count with differential may identify patients with either a malignancy or systemic infection. serologic testing for ebv or cytomegalovirus infection, toxoplasmosis, or cat-scratch disease may be diagnostic. thyroid function testing is essential in any child with a suspected thyroid disorder. finally, collection of urine for catecholamine metabolites (vanillylmandelic acid) may assist in the diagnosis of neuroblastoma. if the diagnosis remains in doubt at this point, incisional or excisional biopsy may be indicated. biopsy provides material for pathologic examination, culture, and other more sophisticated testing if necessary. fine-needle aspiration of a neck mass in children for suspected malignancy is not as reliable as in adults. congenital sinuses and cysts are discussed in chapter 56. viral adenitis is the most common infectious disorder to involve the neck in children. enlarged or hyperplastic lymph nodes are frequently the result of viral upper respiratory tract illnesses. common pathogens include rhinovirus, adenovirus, and enterovirus, but measles, mumps, rubella, varicella, ebv, and cytomegalovirus may also cause lymphadenopathy. the diagnosis is often suspected by other findings in the history or physical examination and can be confirmed by serologic testing. acute human immunodeficiency virus infection may present, as do other viral syndromes, with fever, headache, malaise, gastrointestinal symptoms, and a neck mass. the usual source of bacterial cervical adenitis is the pharynx. causative organisms are often streptococcal or staphylococcal species. patients present with systemic symptoms of fever and malaise in addition to a neck mass that is diffusely swollen, erythematous, and tender. in contrast to viral adenitis, which is frequently bilateral, bacterial infections of the neck are usually unilateral. ct with contrast medium enhancement may be helpful in the evaluation of large infectious neck masses that may contain an abscess cavity. needle aspiration of suspected infectious masses may provide material for culture and decompress the mass. broad-spectrum antibiotic therapy, administered either orally or intravenously, may be curative, although surgical drainage is usually necessary in extensive cases. cat-scratch disease is caused by bartonella henselae infection. the clinical picture includes the sudden appearance of unilateral lymphadenopathy after a scratch from a cat. fever and malaise may be accompanying symptoms in many cases. serologic testing for antibodies to bartonella is diagnostic. cat-scratch disease is usually self-limited, although some benefit has been described with the use of erythromycins and other antibiotics. 21 in the past most mycobacterial infections have been caused by atypical organisms such as mycobacterium avium-intracellulare, m. scrofulaceum, m. bovis, or m. kansasii. in the past decade or so, mycobacterial tuberculosis has made a resurgence as the pathogen responsible for a neck infection. atypical mycobacterial infections present as nontender nodes in the preauricular, intraparotid, or posterior triangle regions. the skin overlying the node typically assumes a violet color, and systemic symptoms are rare. a chest radiograph should be obtained if m. tuberculosis is suspected. the diagnosis is made by obtaining material for acid-fast stain and culture with needle aspiration, surgical drainage, or excision of involved nodes. surgical curettage or total excision is curative for atypical lesions. tuberculosis should be treated with appropriate antituberculin chemotherapy. rarely, the neck may be involved with infections such as tularemia, brucellosis, actinomycosis, plague, histoplasmosis, or toxoplasmosis. inflammatory disorders that may affect the neck include kawasaki syndrome, sarcoidosis, sinus histiocytosis (rosai-dorfman disease), kikuchi-fujimoto disease, and pfapa syndrome (periodic recurrent fever). thyroid malignancies are not uncommon in the adolescent age group, with 10% of thyroid carcinomas occurring in patients younger than 21 years of age. 3 welldifferentiated tumors, usually papillary carcinoma, make up the majority of tumors. follicular, mixed, and medullary tumors occur less commonly. most patients present with a painless midline neck mass. on presentation, cervical adenopathy can be palpated in a majority of patients, a finding that reflects the high incidence of papillary disease that metastasizes via the lymphatics. 9 other important symptoms and signs include a rapid rate of growth, pain, hoarseness, and dysphagia. children who have received prior radiation are at greater risk of thyroid malignancy. the occurrence of thyroid malignancy may be associated with iodine deficiency, hashimoto's thyroiditis, and graves' disease. 9, 22 preoperative assessment should include thyroid nucleotide scanning to distinguish between cold (hypofunctioning) and hot (hyperfunctioning) nodules. up to a third of cold nodules can be malignant, whereas hot nodules are rarely malignant. 12 ultrasonography can distinguish between solid and cystic lesions, and fine-needle aspiration is an alternative to surgical biopsy for diagnosis. surgical management includes near-total or total thyroidectomy, neck dissection if indicated, and postoperative 131 i ablation. lymphoma is a common pediatric malignancy and can present in the neck as painless lymphadenopathy. hodgkin's disease occurs most often in late adolescence and has four histologic subtypes: lymphocyte predominance, nodular sclerosing, mixed cellularity, and lymphocyte depletion. lymphocyte predominance and nodular sclerosing types make up most cases. staging of hodgkin's disease depends on the amount and location of nodal involvement and the presence or absence of systemic or b symptoms (fever, night sweats, weight loss). treatment is with multiple-agent chemotherapy and localized radiation therapy. non-hodgkin's lymphoma can be divided into low-, intermediate-, or high-grade subtypes. high-grade tumors may be further divided into large cell, lymphoblastic, and small cell types. staging of non-hodgkin's lymphoma is by location and extent. treatment is with multiple-agent chemotherapy. langerhans' cell histiocytosis (previously histiocytosis x) includes the disease entities known as eosinophilic granuloma, hand-schã¼ller-christian syndrome, and letterer-siwe disease. the exact nature of this entity remains an enigma: it may represent a neoplasm or a hyperimmune response. 7 symptoms and signs include lymphadenopathy, rashes, otorrhea, oral lesions, and hepatosplenomegaly. diagnosis is dependent on the identification of langerhan's cells on pathologic specimens. treatment ranges from curettage or excision to intralesional or systemic corticosteroids to chemotherapy and radiation therapy. two major categories of neural tumors may be found in the neck. neurofibromatosis is a benign disorder that in some forms (plexiform) may infiltrate surrounding tissues. for this reason, ct and/or mri are vital in the preoperative evaluation of these lesions. surgical resection is the mainstay of treatment. neuroblastoma is a malignancy that develops from neural crest cells and may present as a solitary tumor or as lymphadenopathy. clinical staging determines the mode of therapy that includes surgery, chemotherapy, and radiation therapy. rhabdomyosarcoma rarely presents as a primary tumor in the neck, more often being found as a primary tumor in the orbit, temporal bone, or nasopharynx. the diagnosis is made by biopsy, and patients are staged according to involvement. treatment includes surgery, chemotherapy, and radiation therapy. malignancies of almost any type and location in the body can metastasize to the neck. the most common are thyroid malignancies. in adolescents, carcinomas, especially those arising in the nasopharynx, may spread to the neck lymphatics. anatomy and embryology of the paranasal sinuses otitis media and eustachian tube dysfunction cancer of the thyroid in youth pediatric malignancies neoplasms of the ear and temporal bone congenital malformations of the temporal bone head and neck langerhans' cell histiocytosis otolaryngologic disorders lymphomas of the head and neck thyroid tumors in children corticosteroids in airway management allergic fungal sinusitis: pathophysiology, epidemiology and diagnosis solitary thyroid nodules in 71 children and adolescents glomus tympanicum in infancy grading system for the selection of patients with congenital aural atresia an evidence-based review of the treatment of peritonsillar abscess steroid treatment of laryngotracheitis: a meta-analysis of the evidence from randomized trials childhood epiglottitis in recent years is fetal cellular rhabdomyoma an entity or a differentiated rhabdomyosarcoma? a study of patients with rhabdomyoma of the tongue and sarcoma of the tongue enrolled in the intergroup rhabdomyosarcoma studies i, ii and iii squamous cell carcinoma of the tongue in a nine-year renal transplant survivor: a case report with a discussion of the risk of development of epithelial carcinoma in renal transplant survivors normal polysomnographic values for children and adolescents current knowledge of bartonella species thyroid carcinoma in children and adolescents pediatric audiology efficacy of tonsillectomy for recurrent throat infection in severely affected children: results of parallel randomized and nonrandomized clinical trials first-line treatment of otitis media structure and function of the temporal bone surgical pediatric otolaryngology influence of penicillin-producing staphylococci and the eradication of group a streptococci from the upper respiratory tract by penicillin treatments angiofibroma: changes in staging and treatment regional and intracranial complications of sinuses pediatric neck masses: guidelines for evaluation computed tomography in the evaluation of pediatric neck infections pediatric cochlear implantation key: cord-349287-mwj2qby4 authors: mackay, ian m.; arden, katherine e. title: mers coronavirus: diagnostics, epidemiology and transmission date: 2015-12-22 journal: virol j doi: 10.1186/s12985-015-0439-5 sha: doc_id: 349287 cord_uid: mwj2qby4 the first known cases of middle east respiratory syndrome (mers), associated with infection by a novel coronavirus (cov), occurred in 2012 in jordan but were reported retrospectively. the case first to be publicly reported was from jeddah, in the kingdom of saudi arabia (ksa). since then, mers-cov sequences have been found in a bat and in many dromedary camels (dc). mers-cov is enzootic in dc across the arabian peninsula and in parts of africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. the ksa is the focal point of mers, with the majority of human cases. in humans, mers is mostly known as a lower respiratory tract (lrt) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. however, mers-cov has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. older males most obviously suffer severe disease and mers patients often have comorbidities. compared to severe acute respiratory syndrome (sars), another sometimesfatal zoonotic coronavirus disease that has since disappeared, mers progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. most human cases of mers have been linked to lapses in infection prevention and control (ipc) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (hcws) and higher exposures in those with occupations that bring them into close contact with camels. sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics have been available almost from the start of the emergence of mers. while the basic virology of mers-cov has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. electronic supplementary material: the online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. an email from dr ali mohamed zaki, an egyptian virologist working at the dr soliman fakeeh hospital in jeddah in the kingdom of saudi arabia (ksa) announced the first culture of a new coronavirus to the world. the email was published on the website of the professional emerging diseases (promed) network on 20 th september 2012 [1] (fig. 1) and described the first reported case, a 60 year old man from bisha in the ksa. this information led to the rapid discovery of a second case of the virus, this time in an ill patient in the united kingdom, who had been transferred from qatar for care [2] . the new virus was initially called novel coronavirus (ncov) and subsequentlty entitled the middle east respiratoy syndrome coronavirus (mers-cov). as of 2 nd of september 2015, there have been 1,493 detections of viral rna or virus-specific antibodies across 26 countries (additional file 1: figure s1 ) confirmed by the world health organization (who), with over a third of the positive people dying (at least 527, 35 %) [3] . since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (dc; camelus dromedarius) in the ksa [4] , also dcs across the arabian peninsula and parts of africa that are a source of dc imports for the ksa [5] . to date, mers-cov has not been detected in dcs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . occasionally, virus is transmitted from infected dcs to exposed humans. subsequent transmission to other humans requires relatively close and prolonged exposure [10] . the first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . however, sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (dpp4; also called cd26); that mers-cov has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than sars-cov; is widespread in dcs; has the potential to infect other animals and that mers kills its human host more often than sars did (20-40 % versus 9 % for sars [14] ) [15] [16] [17] [18] [19] . in humans, overt disease was given the name middle east respiratory syndrome, with the acronym mers. from intermittent animal-to-human spill-over events, the mers-cov spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. the spread of mers-cov among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (hcws). although dcs appear to suffer the equivalent of a 'common cold' from mers-cov infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. it has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . studies have established that the mean incubation period for mers is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . the first who case definition [27] defined probable cases of mers based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (lrt) involvement. it also included roles for contact with a probable or confirmed case or for travel or residence within the arabian peninsula. if strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . from july 2013, the revised who case definition included the importance of seeking out and understanding the role of asymptomatic cases and from june 2014, the who definition more clearly stated that a confirmed case included any person whose sample was rt-pcr positive for mers-cov, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] apart from the who and the ksa ministry of health reports, asymptomatic or subclinical cases of mers-cov infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . the ksa definition of a case became more strict on 13 th may 2014, relying on the presence of both clinical features and laboratory confirmation [33] . testing of asymptomatic people was recommended against from december 2014 [34] , reinforced by a case definition released by the ksa ministry of health in june 2015 [35] . the ksa has been the source of 79 % of human cases. severe mers is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . interestingly in june 2015, an outbreak in south korea followed a similar distribution [39, 40] . among laboratory confirmed cases, fever, cough and upper respiratory tract (urt) signs and symptoms usually occur first, followed within a week by progressive lrt distress and lymphopaenia [37] . patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . mers has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the ksa, yet only 19 % of cases in south korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . general supportive care is key to managing severe cases [43] . children under the age of 14 years are rarely reported to be positive for mers-cov, comprising only 1.1 % (n = 16) of total reported cases. between 1 st september 2012 and 2 nd december 2013, a study described the then tally of paediatric cases in the ksa, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . in amman, jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for mers-cov rna, despite being collected at a similar time to the first known outbreak of mers-cov in the neighbouring town of al-zarqa [45] . a second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not rt-rtpcr positive, the mother did subsequently develop antibodies to mers-cov, suggestive of recent infection [46] . her exposure history to a mers-cov rt-rtpcr positive relative and an antibody-reactive husband, her incubation period and her symptom history met the who criteria for being a probable mers-cov case [46] . diagnostic methods were published within days of the promed email announcing the first mers case [47] , including several now gold standard in-house rt-rtpcr assays (fig. 2 ) as well as virus culture in vero and llc-mk2 cells [18, 47, 48] . a colorectal adenocarcinoma (caco-2) epithelial cell line has since been recommended for isolation of infections mers-cov [49] . we previously [18] .). open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (utr; grey rectangles). fs-frame-shift. predicted regions encompassing recombination break-points are indicated by orange pills. created using geneious v8.1 [211] and annotated using adobe illustrator. beneath this is a schematic depicting the location of rt-pcr primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest rt-rtpcr screening assays and conventional, semi-nested (three primers) rt-pcr confirmatory sequencing assays [47, 48] . publication order is noted by first [27 th september 2012; red] and second [6 th december 2012; orange] coloured rectangles; both from corman et al. [47, 48] those assays recommended by the who are highlighted underneath by yellow dots [53] . the nseq reverse primer has consistently contained one sequence mismatch with some mers-cov variants. an altered version of that from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res 2015 vol 202:60-88 with permission from elsevier [5] reviewed the broad tropism of mers-cov [5] . however, as is well described, cell culture is a slow, specialised and insensitive method [50] while pcr-based techniques are the preferred method for mers-cov detection. the first open reading frames (orf 1a and 1b; fig. 2 ) have become a key diagnostic and taxonomic target for cov species identification. with less than 80 % identity between the amino acid sequence of mers orf 1ab and betacoronavirus relatives, tylonycteris bat hku4 and pipistrellus bat hku5, it can be concluded that it is a novel and distinct virus. mers-cov is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . the structural proteins include the spike (s), envelope (e), membrane (m) and nucleocapsid (n) [52] . the products of orf1a and orf1b are predicted to encode nonstructural proteins. the majority of specimen testing to date has employed validated rt-rtpcr assays shown to be sensitive and specific [47, 48, 53] . the realstar® kit uses these whorecommended assays [54] . the target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (imm observation). other rt-rtpcr assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. the detection of mers-cov antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. detection of viral proteins rather than viral rna indicates the likely presence of infectious virus. the first rapid immunochromatographic tool described could detect recombinant mers-cov nucleocapsid protein from dc nasal swabs with 94 % sensitivity and 100 % specificity compared to rt-rtpcr [61] . a different approach used a monoclonal antibody-based capture elisa targeting the mers-cov nucleocapsid protein with a sensitivity of 10 3 tcid 50 and 100 % specificity [62] . demonstration of a seroconversion to a mers-cov infection meets the current who definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior mers-cov infection and help support transmission studies. because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral rna [63] . strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. much of the arabian peninsula and all of the horn of africa lack baseline data describing the proportion of the community who may have been infected by a mers-cov. however, sero-surveys have had widespread use in elucidating the role of dcs as a transmission source for mers-cov. because of the identity shared between dc and human mers-cov (see molecular epidemiology: using genomes to understand outbreaks), serological assays for dc sero-surveys should be transferrable to human screening with minimal re-configuration. also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested mers-cov isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single mers-cov serotype [49] . the development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to mers-cov, as well as to likely sources of cross-reaction [64] . obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . a number of commercial elisa kits, immunofluorescent assays (ifa) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . initially, conventional ifas were used for human sero-surveys. these relied on mers-cov-infected cell culture as an antigen source, detecting the presence of human anti-mers-cov igg, igm or neutralizing antibodies in human samples [18, 48, 69] . no sign of mers-cov antibodies was found among 2,400 sera from patients visiting hospital in jeddah, from 2010 through 2012, prior to the description of mers-cov [18] . nor did ifa methods detect any sign of prior mers-cov infection among a small sample of 130 healthy blood donors from another hospital in jeddah (collected between jan and dec 2012) [70] . of 226 slaughterhouse workers, only eight (3.5 %) were positive by ifa, and those sera could not be confirmed by virus neutralization (nt) test. the study indicated that hcov-hku1 was a likely source of crossreactive antigen in the whole virus ifa [70] . whole virus mers-cov ifa also suffered from some cross-reactivity with convalescent sars patient sera and this could not be resolved by an nt test which was also cross-reactive [71] . ifa using recombinant proteins instead of whole-virus ifa, has been shown to be a more specific tool [31] . since asymptomatic zoonoses have been posited [72] , an absence of antibodies to mers-cov among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering dcs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. ifa using recombinant proteins instead. some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the mers-cov nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing mers-cov spike protein and luciferase [74, 75] . a pseudo particle neutralization (ppnt) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (mnt) test. [10, 74, [76] [77] [78] ] studies using small sample numbers and ppnt found no evidence of mers-cov neutralizing antibody in sera from 158 children with lrt infections between may 2010 and may 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the jazan region of the ksa during 2012 [79, 80] . similarly, a study of four herdsmen in contact with an infected dc herd in al-ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in al-ahsa and 146 controls who were not exposed to dcs in any professional role, found none with serological evidence of past mers-cov infection using the ppnt assay [10] . a delay in the neutralizing antibody response to mers-cov infection was associated with increased disease severity in south korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . the implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. a jordanian outbreak of acute lrt disease in a hospital in 2012 was retrospectively found to be associated with mers-cov infection, initially using rt-rtpcr, but subsequently, and on a larger scale, through positivity by elisa and ifa or mnt test. [46, 82, 83] this outbreak predated the first case of mers in the ksa. the elisa used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-cov hku5 to identify antibodies against the equivalent crossreactive mers-cov protein [71] . it was validated using 545 sera collected from people with prior hcov-oc43, hcov-229e, sars-cov, hcov-nl63, hrv, hmpv or influenza a(h1n1) infections but was reportedly less specific than the recombinant ifa discussed above. it was still considered an applicable tool for screening large sample numbers [82] . a protein microarray expressing the s1 protein subunit has also been validated and widely used for dc testing [5, 84] . detection of mers-cov infection using elisa or s1 subunit protein microarray [84] is usually followed by confirmatory ifa and/ or a plaque-reduction neutralization (prnt) [69, 70, 85] or mnt test. [74, 85, 86] this confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in dcs (bovine cov, bcov) or humans (hcov-oc43, hcov-229e, hcov-nl63, hcov-hku1, sars-cov). in the largest study of human sera, a tiered diagnostic process assigned both recombinant ifa and recombinant elisa positive sera to 'stage 1' seropositivity. a stage 2 seropositive result additionally required a suitably titred prnt result [87] . the study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 ksa provinces contained mers-cov antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . contemporary surveys are needed. mers-cov does not appear to be easily transmitted from dcs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. this was reinforced when a false positive us case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, nt assay and was subsequently retracted [88, 89] . the who recommends sampling from the lrt for mers-cov rt-rtpcr testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] lrt samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . recommended sample types include bronchoalveolar lavage (bal), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°c [90] . if available, lung biopsy or autopsy tissues can also be tested [53] . the urt is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when urt sampling is to be conducted [90] . paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting rt-rtpcr [53, 90] . human urine and stool have been found to contain mers-cov rna 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . in two cases that arrived in the netherlands, urine was rt-rtpcr negative but faeces was weakly positive and sera were rt-rtpcr positive for five days or more [25] . the finding of mers-cov viral rna in serum provides an avenue for retrospective pcr-based studies if respiratory samples are unavailable [83] . rnaaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . clinically suspected mers cases may return negative results by rt-rtpcr. data have shown one or more negative urt samples may be contradicted by further urt sampling or the use of lrt samples, which is preferred [2, 43, 93] . higher viral loads occur in the lrt compared to the urt. [22, 69, 88, 94] this fits with the observation that the majority of disease symptoms are reported to manifest as systemic and lrt disease [21] . however, on occasion, even lrt specimens from mers cases may initially be negative, only to later become positive by rt-pcr [95] . this may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . despite this both the largest human mers-cov studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the urt. it is then noteworthy that one study reported an association between higher loads in the urt and worse clinical outcome including intensive care and death [94] . at writing, no human data exist to define whether the virus replicates solely or preferentially in the lrt or urt, or replicates in other human tissues in vivo although mers-cov rna has been detected from both the urt and lrt in a macaque monkey model [100] .the distribution of dpp4 in the human upper airways is also not well described. individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] in one instance, a hcw shed viral rna for 42 days in the absence of disease [99] . it is an area of high priority to better understand whether such cases are able to infect others. over three quarters of mers cases shed viral rna in their lrt specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding rna in their urt specimens [91, 102] . in the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (npa; an urt sample), 30 tracheal aspirates, 13 sputa and three bal were examined. the tracheal aspirates and bal returned the highest viral load values followed by npa and sputum. unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in npa testing, were significantly correlated with severe disease and death [49, 94, 103] . this study demonstrated the importance of lrt sampling for whole genome sequencing. when tested, samples positive for mers-cov are often negative for other pathogens [2, 25, 93, 104] . however, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . when viruses are sought, they have included human herpesvirus (hhv), rhinoviruses (hrv), enteroviruses (ev), respiratory syncytial virus (rsv), parainfluenzavirus types 1, 2 and 3 (pivs),influenzaviruses (ifvs), endemic hcovs, adenoviruses (advs) metapneumovirus (mpv) and influenza a\h1n1 virus; co-detections with mers-cov have been found on occasion [2, 22, 37, 69, 97] . bacterial testing is sometimes included (for example, for legionella and pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . further testing of the lrt sample from the first mers case used ifa to screen for some viruses (negative for ifv, pivs, rsv and advs) and rt-pcr for others (negative for adv, evs, mpv and hhvs) [18] . rt-pcr also detected mers-cov. the who strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both mers cases and their contacts. little is known of other causes of mers-like pneumonia in the ksa or of the general burden of disease due to the known classical respiratory viruses. testing of adult pilgrims performing the hajj in 2012 to 2014 has not detected any mers-cov. in 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the ksa were tested [47] . in 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . it should be noted that most pilgrims arrived from mers-free countries. a further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . in earlier hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among hajj pilgrims. [110] a lrt sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of mers-cov infection and replication; it may have been assumed it was the lrt because disease was first noticed there but the urt may be the site of the earliest replication. in jeddah between march and july 2014 (hereafter called the jeddah-2014 outbreak; fig. 3 ), there was a rapid increase in mers cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 mers-cov detections (~3 % prevalence) [111] . among 5,065 individuals sampled and tested across the ksa between october 2012 and september 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated hcws (n = 1,695; 33.5 %) [32] . among the detections, 19 (17.8 %) were hcws and 10 (9.3 %) were family contacts [32] . the 2-3 % prevalence of active mers-cov infections is not dissimilar to the hospital-based prevalence of other human covs. [112] however, the proportion of deaths among those infected with mers-cov is much higher than that known for the hcovs nl63, hku1, 229e or oc43 in other countries, and even above that for sars-cov; it is not a virus that could reasonably be described as a "storm in a teacup". it is the low transmission rate that has prevented worldwide spread, despite many "opportunities". very early in the mers outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of mers-cov with three of the first five cases having contact with dcs [73, 113, 114] . today, animal mers-cov infections must be reported to the world organization for animal health as an emerging disease [115] . a summary of the first mers cases reported by the who defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . this definition made no specific allowance for acquisition from dcs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . camels are known to produce high levels of mers-cov rna in their urt and lungs [116] . providing support for a droplet transmission route and perhaps indicating the presence of rna in smaller, drier droplet nuclei, mers-cov rna was identified in a high volume air sample collected from a barn housing an infected dc [117] . the precise source from which humans acquire mers-cov remains poorly studied but it seems likely that animal and human behavioural factors may play roles (fig. 3) [118] . these factors may prove important for human cases who do not describe any dc contact [119] nor any contact with a confirmed case. whether the who definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. wording focuses on consumption of dc products but does not specifically ascribe risk to a droplet route for acquisition of mers-cov from dc [120] . some mers patients are listed in who disease notices as being in proximity to dcs or farms, but the individuals have not described coming into contact with the animals. no alternative path for acquiring infection is reported in many of these instances. what constitutes a definition of "contact" during these interviews has been defined for one study [72] . despite this lack of clarity, the who consider that evidence linking mers-cov transmission between dcs to humans is irrefutable (fig. 4) [120] . the possibility that bats were an animal host of mers-cov was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . conclusive evidence supporting bats as a source for human infections by mers-cov has yet to be found, but bats do appear to host ancestral representatives [53, 125] . however, these are not variants of the same virus nor always within the same phylogenetic lineage as mers-cov; they are each a genetically distinct virus. bat-to-human infection by mers-cov is a purely speculative event. the only piece of mers-cov-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the rnadependent rna polymerase gene of the mers-cov genome, identified in a faecal pellet from an insectivorous emballonuridae bat, taphozous perforatus found in bisha, the ksa [121] . while very short, the sequence of the fragment defined it as a diagnostic discovery. subsequently a link to dcs was reported [85] and that link has matured into a verified association [38, 126] (fig. 4) . (see figure on previous page.) fig. 3 monthly detections of mers-cov (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th september 2015. an approximation of when dc calving season [128] and when recently born dcs are weaned is indicated. spring (green) and summer (orange) in the arabian peninsula are also shaded. note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. sources of these public data include the who, ministries of health and flutrackers [207] [208] [209] . earlier and subsequent versions of this chart are maintained on a personal blog [210] . modified and reprinted from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res 2015 vol 202:60-88 with permission from elsevier [5] dcs, which make up 95 % of all camels, have a central presence in the arabian peninsula where human-dc contact ranges from little to close [119] . contact may be commonplace and could occur in variety of ways (fig. 4a) . there are several large well-attended festivals, races, sales and parades which feature dcs and dcs are also kept and bred close to populated areas in the ksa [127, 128] . dc milk and meat are widely consumed and the older dc is an animal of ritual significance after the hajj pilgrimage [129] . however, mers-cov infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing dc products. daily ingestion of fresh unpasteurized dc milk is common among the desert bedouin and many others in the ksa. dc urine is also consumed or used for supposed health benefits. despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among mers cases; this may simply be a reporting issue rather than an unexplainable absence of mers. a small case-control study published in 2015 identified direct dc contact, and not ingestion of products, to be associated with onset of mers [38] . the first sero-survey of livestock living in the middle east region was conducted during 2012-2013 [85] . dcs were sampled from a mostly canary island-born herd and from omani dcs (originally imported from the horn of africa) [85] . a neutralising antibody assay found only 10 % of strongly seropositive canary island [5] . b camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor ipc in healthcare settings where transmission is amplified, accounting for the bulk of cases. there are human mers cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) mers-cov infection may be implicated in transmission dc sera could neutralise mers-cov while all omani dc sera had high levels of specific mers-cov neutralizing antibody [85] . this indicated that dcs had in the past been infected by mers-cov, or a very similar virus. since this study, a host of peer-reviewed reports have looked at both dcs and other animals, and the possibility that they may host mers-cov infection. seropositive dcs have been found throughout the arabian peninsula including oman, the ksa, qatar, jordan, the united arab emirates (uae), kuwait as well as sudan, somalia, egypt, tunisia, nigeria, kenya and ethiopia in africa and the canary islands [85, [130] [131] [132] [133] [134] . other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, bactrian camels, llamas and guanaco (south american camelids) but none had detectable neutralising antibody against mers-cov [4, 74, 78, 85, 86, 135, 136] . no virology or serology studies of human samples from areas in africa where there are camels with a history of mers-cov have been reported to date. however,an absence of unexplained pneumonia that may be attributable to mers-cov infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. it is thus unclear whether mers-cov, or an antigenically related cov, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the arabian peninsula [133] . mers-cov rna has also been detected in dc samples, and recovery of infectious virus has also been achieved from dc samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . from some of these, full or majority length genomes of mers-cov have been sequenced [77, 137, 138] . dc versions of mers-cov were found to be as similar to each other, as were variants detected from different humans over time and across distance. antibody screening assays have also detected crossreactive antibodies in sera. these were identified as such by screening sera against similar viruses, for example bcov or hcov-oc43 (as an antigenic facsimile for bcov). it is possible that other mers-cov-like viruses also reside within dcs, but this does not detract from the definitive finding of mers-cov genetic sequences in both dcs and humans [117, 142, 143] . screening studies have shown that juvenile dcs are more often positive for virus or viral rna while older dcs are more likely to be seropositive and rna or virus negative [76, 77, 144] . in adult dcs, mers-cov rna has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . viral loads among positive dcs can be very high [4, 76, 77, 139, 144] and dcs have been found positive both when ill with urt respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . these findings indicate dcs host natural mers-cov infections. furthermore, stored dc sera have revealed signs of mers-cov in dcs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . older sera have not been tested and so precisely how long dcs have been afflicted by mers-cov, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in africa or the arabian peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. researchers sought to determine a direction for infection; were dcs transmitting virus to humans or were humans infecting dcs? at a qatari site, a farm owner and his employee became ill in mid-october 2013 and tested positive for mers-cov rna in a sputum and throat swab sample, respectively. rt-rtpcrs found mers-cov rna in 11 of 14 positive dc nasal swabs at the farm; six (43 %) positive by two or more assays [138] . the results indicated a recent outbreak had occurred in this herd; the first indication of mers-cov rna found within dcs with a temporal association to human infections. three positive dc samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and dc mers-cov sequences [138] . the dcs and human contacts yielded orf1a and orf4b sequences differing by only a single nucleotide each, clustering closely with the hafr-al-batin_1_2013 variant [138] . subsequent case studies found evidence of a concurrent human and dc infection and the direction of that infection was inferred to be from the ill dcs and to their human owners [117, 142, 146] . partial genome sequences indicated that a human and a mers-cov rt-rtpcr positive dc had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] all nine dc in the owner's herd, serially sampled, reacted in a recombinant s1 antigen elisa, with the two animals that had been rt-rtpcr positive showing a small, verifiable rise in antibody titre [142] . a rise in titre theoretically begins 10 to 21 days after dc infection [142] . the authors suggested that the rise in titre in dc sera which occurred alongside a declining rna load, while the patient was actively ill and hospitalized, indicated that the dcs were infected first followed by the owner [117, 142] . bcov antibodies were also present, and rising in one of the two rt-rtpcr positive animals but no animal's antibodies could neutralise bcov infection [142] . camel calving season occurs in the winter months (between late october and late february; fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve dc populations [128] . what role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . juvenile dcs appear to host active infection more often than adult dcs and thus the sacrificial slaughter of dcs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. in contrast to earlier results, slaughterhouse workers who kill both younger and older dcs, may be an occupational group with significantly higher incidence of seropositivity to mers-cov when animals have active mers-cov infections [129, 139, [147] [148] [149] . expanded virological investigations of african dcs may lead to more seropositive animals and geographic areas in which humans may be at risk. it is possible that there are areas where humans already harbour mers-cov infections that have not been identified because of an absence of laboratory surveillance. virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . infectious mers-cov added to dc, goat or cow milk and stored at 4°c could be recovered at least 72 h later and, if stored at 22°c, recovery was possible for up to 48 h [150] . mers-cov titre decreased somewhat when recovered from milk at 22°c but pasteurization completely ablated mers-cov infectivity [150] . in a subsequent study, mers-cov rna was identified in the milk, nasal secretion and faeces of dcs from qatar [151] . a single study has examined the ability of mers-cov to survive in the environment [150] . plastic or steel surfaces were inoculated with 10 6 tcid 50 of mers-cov at different temperature and relative humidity (rh) and virus recovery was attempted in cell culture. at high ambient temperature (30°c) and low rh (30 %) mers-cov remained viable for 24 h [150] . by comparison, a well known and efficently transmitted respiratory virus, influenza a virus, could not be recovered in culture beyond four hours under any conditions [150] . aerosol experiments found mers-cov viability only decreased 7 % at low rh at 20°c. in comparison, influenza a virus decreased by 95 % [150] . mers-cov survival is inferior to that previously demonstrated for sars-cov [152] . for context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. mers-cov's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . whether mers-cov can remain adrift and infectious for extended periods (truly airborne) remains unknown. such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. the nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the al-ahsa hospital, the ksa and the south korean outbreaks [21, 23, 154, 155] . by extrapolation, aerosol-generating events involving dcs (urination, defecation, and preparation and consumption of dc products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. the provision of evidence supporting the best formulation of personal protective equipment to be worn by hcws who receive, manage or conduct procedures on infectious cases remains a priority. mers-cov was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to mers-cov transmission chains. however, transmission of mers-cov is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] in a household study, 14 of 280 (5 %) contacts of 26 mers-cov positive index patients were rna or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . it seems that the majority of human cases of mers-cov, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of mers-cov has not been self-sustaining [157] [158] [159] [160] [161] . that is to say, the basic reproduction number (r 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. if r 0 was greater than 1, a sustained increase in case numbers would be expected. some r o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. that mers has had a constant presence in the arabian peninsula since 2012 is due to ongoing, sporadic spill-over events from dcs amplified by poorly controlled hospital outbreaks. the first known mers human-to-human transmission event was one characterized by acute lrt disease in a healthcare setting in jordan. in stark contrast, a sero-survey of hcw who were sometimes in close and prolonged contact with the first, fatal mers-cov case in 2012 [162] , found none of the hcw had seroconverted four months later, despite an absence of eye protection and variable compliance with required ppe standards [162] . early on in the mers story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. contacts of confirmed mers cases were often observed for clinical illness, but not tested. these omissions may have confounded our understanding of mers-cov transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. case-control studies were not a focus. as testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . a rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the jeddah-2014 outbreak. historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. upon follow-up, over three-quarters of such mers-cov rna positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. the proportion of fatal mers cases within the ksa compared to outside the ksa, as well as the age, and sex distribution change in different ways when comparing mers outbreaks. approximately 43 % of mers cases (549 of 1277) in the ksa were fatal betwen 2012 and december 2015 while 21 % (72 of 330) died among those occurring outside of the ksa. the total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. however the proportion of male deaths from total males with mers is a similar figure to that for females. in the ksa, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 south korean or the jeddah-2014 outbreaks (additional file 2: figure s2 ). why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . as a group, hcws comprised 16 % of mers cases in the ksa and south korea. it is apparent that the weekly proportion of infected hcws increases alongside each steep rise in overall detections (fig. 5) . in may 2013, the who published guidelines for ipc during care of probable or confirmed cases of mers-cov infection in a healthcare setting [166] . this is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . these rises in mers-cov detections can decrease the average age during each event because hcws are usually younger than inpatients with mers. healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (ipc) procedures [115, 118] . most of the analysis of mers-cov genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . mers-cov was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. the technique can produce genomic [207] [208] [209] . earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during mers-cov characterization [48] . as more genomes from both humans and dcs have been characterized, two clades have become apparent; a and b (fig. 6) . clade a contains only human-derived mers-cov genomes from jordan, while clade b comprises the majority of human and camel genomes deduced thus far [168] . two studies during 2015, one looking at jeddah-2014 mers-cov variants and another looking at a variant exported from south korea to china, have now identified signs of genetic recombination among mers-cov variants. while human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . shared identity implies that the major source for human acquisition is the dc, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. over a month, a dc virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that dcs are the natural, rather than intermediate, host for the mers-cov we know today [77] . to date, recombination has been localised to breakpoints near the boundary between orf1a and orf1b regions, within the spike gene [170] and in the orf1b region (fig. 2) [172] . it is not unexpected that recombination should occur since it is well known among other covs [124] and because the majority of mers-cov whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. if we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely fig. 6 the genetic relationship between mers-cov nucleotide sequences (downloaded from genbank using the listed accession numbers and from virological.org [212] ). this neighbour joining tree was created in mega v6 using an alignment of human and dcderived mers-cov sequences (geneious v8.1 [211] ). clades are indicated next to dark (clade a) or pale (clade b) blue vertical bars. camel icons denote genomes from dcs. healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. genetic mutations noted during the largest of human outbreaks, jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . however, we understand very little of the phenotypic outcomes that result from subtle genetic change in mers-cov genomes. to date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . but vigilance and larger, more contemporary and in vivo studies are needed. genome sequence located to a distinct clade were identified from an egyptian dc that was probably imported from sudan. this does not fit into either of the current clades [125, 168, 177] . a virus sequenced from a neoromicia capensis bat was more closely related to mers-cov than other large bat-derived sequences had been to that point, but the genome of a variant of a mers-cov has yet to be discovered and deduced from any bat [125] . analyses of mers-cov genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (fig. 2) , which encodes the spike protein and accessory proteins [168] . at least nine mers-cov genomes contained amino acid substitutions in the receptor binding domain (rbd) of the spike protein and codons 158 (n-terminal region), 460 (rbd), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . the spike protein had not changed in the recombinant mers-cov genome identified in china in 2015 but was reported to have varied at a higher rate than that for complete mers-cov genomes, among south korean variants [172, 178] . this highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. despite this, one assay amplifying a 615 nucleotide fragment of the spike s2 domain gene for sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (fig. 6 ) [169] . this approach was employed when defining the geographically constrained mers hospital outbreak in al-ahsa, which occurred between 1 st april and 23 rd may 2013, as well as clusters in buraidah and a community outbreak in hafr al-batin, the ksa. genomic sequencing identified that approximately 12 mers-cov detections from a community outbreak in hafr al-batin between june and august 2013 may have been triggered by an index case becoming infected through dc contact [175] . sequencing mers-cov genomes from the 2013 al-ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . however, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . in riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . riyadh is a nexus for camel and human travel and has had more mers cases than any other region of the ksa to date but also harbours a wide range of mers-cov variants [128, 167, 179] . however the south korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . for many mers cases detected outside the arabian peninsula, extensive contact tracing has been performed and the results described in detail. contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. results of contact tracing to date have found that onward transmission among humans is an infrequent event. for example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in germany who travelled from the uae but no sign of virus or antibody were found in any of them [73] . the very first mers case had made contact with 56 hcws and 48 others, but none developed any indication of infection [162] . in a study of 123 contacts of a case treated in france, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . none of the contacts of the first two mers cases imported into the usa in 2014 contained any mers-cov footprint [182] and none of the 131 contacts of two travellers returning to the netherlands developed mers-cov antibodies or tested rna positive [25, 183] . analyses of public data reveal many likely instances of nosocomial acquisition of infection in the arabian peninsula and these data may be accompanied by some details noting contact with a known case or facility. one example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple south korean hospitals [184] . such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (fig. 4c) . the hospital-associated outbreak in jeddah in 2014 was the largest and most rapid accumulation of mers-cov detections to date. the greatest number of mers-cov detections of any month on record occurred in jeddah in april. the outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . a rise in fatalities followed the rapid increase in case numbers. in 2015 two large outbreaks occurred. south korea was the site of the first large scale outbreak outside the arabian peninsula and produced the first cases in both south korea and china, occurring between may and july 2015. this was closely followed by a distinct outbreak in ar riyad province in the ksa which appeared to come under control in early november. after staying in bahrain for two weeks, a 68 year old male (68 m) travelled home to south korea via qatar, arriving free of symptoms on the 4 th may 2015 [187] . he developed fever, myalgia and a cough nearly a week later (11 th ). he visited a clinic as an outpatient between the 12 th and 15 th of may and was admitted to hospital a on the 15 th [188] . he was discharged from hospital a on the 17 th then visited and was admitted to the emergency department of hospital b on the 18 th . during this second stay, a sputum sample was taken and tested positive for mers-cov on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the south korean outbreak. approximately 34 people were infected during this time [187] . in total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 m; 37 cases died [189] . in south korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with mers, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of korean society, crowded emergency rooms, poor ipc measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . all of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. the hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . early in the outbreak, a infected traveller, the son of an identified case in south korea, passed through hong kong on his way to china where he was located, isolated and cared for in china [91, 199, 200] . no contacts became ill. the outbreak was brought under control in late july/ early august [201] after improved ipc measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . a review of public data showed that, as for mers in the ksa, older age and the presence of underlying disease were significantly associated with a fatal outcome in south korea. [40] even though r 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from mers-cov infection [204] . the dynamic of an outbreak depends on the r 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . in the region of ar riyad, including the capital city of riyadh, a hospital based cluster began, within a single hospital, from late june 2015 [205] . by mid-september there had been approximately170 cases reported but the outbreak appeared to been brought under control in november. it became apparent early on that mers-cov spread relatively ineffectively from human-to-human. despite ongoing and possibly seasonal introduction of virus to the human population via infected dcs and perhaps other animals yet to be identified, the vast majority of mers-cov transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. this opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. it remains unclear if this group are uniquely affected by mers-cov or if other respiratory virus infections, including those from hcovs, produce a similarly serious impact. in south korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. household human-to human transmission occurs but is also limited. educational programs will be essential tools for combatting the spread of mers-cov both within urban and regional communities and for the health care setting. vigilance remains important for containment since mers-cov is a virus with a genetic makeup that has been observed for only three years and is not stable. among all humans reported to be infected, nearly 40 % have died. continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. whole genome sequencing has been used extensively to study mers-cov travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. mers and sars have some clinical similarities but they also diverge significantly [206] . defining characteristics include the higher pfc among mers cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of sars) and the higher association between fatal mers and older males with underlying comorbidities. for the viruses, mers-cov has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to sars-cov. there appears to be a 2-3 % prevalence of mers-cov in the ksa with a 5 % chance of secondary transmission within the household. there is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any r 0 would predict on face value. nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of mers or mers-cov during or immediately after these events. there is no evidence that mers-cov is a virus of pandemic concern. nonetheless, hospital settings continue to describe mers cases and outbreaks in the arabian peninsula. as long as we facilitate the spread of mers-cov among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. the mers-cov appears to be an enzootic virus infecting the dc urt with evidence of recent genetic recombination. it may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. rt-pcr testing of lrt samples remains the gold standard for mers-cov confirmation. serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. similarly, the important question of whether those who do shed mers-cov rna for extended periods are infectious while appearing well, continues to go unanswered. it is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. while the basic virology of mers-cov has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. additional file 1: figure s1 . the severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd absence of mers-coronavirus in bactrian camels, southern mongolia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome coronavirus infection not found in camels in japan absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread lack of middle east respiratory syndrome coronavirus transmission from infected camels tensions linger over discovery of coronavirus as outbreak continues middle east respiratory syndrome coronavirus (mers-cov): evidence and speculations cumulative number of reported probable cases of sars in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection replicative capacity of mers coronavirus in livestock cell lines human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-tohuman transmission case definition for case finding severe respiratory disease associated with novel coronavirus middle east respiratory syndrome coronavirus case definition for reporting to who. interim case definition as of 14 middle east respiratory syndrome coronavirus | case definition for reporting to who 14 revised interim case definition for reporting to who -middle east respiratory syndrome coronavirus (mers-cov): interim case definition as of transmission of mers-coronavirus in household contacts screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study case definition and surveillance guidance for mers-cov testing in saudi arabia infection prevention/control and management guidelines for patients with middle east respitaory syndrome coronavius (mers-cov) infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection middle east respiratory syndrome coronavirus in children characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia preliminary epidemiological assessment of mers-cov outbreak in south korea mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea invited editorial: mers-cov an emerging viral zoonotic disease: three years after and counting middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in stillbirth during infection with middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia development and evaluation of a 'real-time' rt-pcr for the detection of enterovirus and parechovirus rna in csf and throat swab samples mers: emergence of a novel human coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans laboratory testing for middle east respiratory syndrome coronavirus | interim guidance performance and clinical validation of the realstar mers-cov kit for detection of middle east respiratory syndrome coronavirus rna real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus laboratory testing for middle east respiratory syndrome coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses realtime sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) a sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in serological assays for emerging coronaviruses: challenges and pitfalls middle east respiratory syndrome coronavirus immunoassays test characteristics: anti-mers-cov elisa camel (igg) recombivirus anti-middle east respiratory syndrome coronavirus (mers-cov) elisa kits ncov / novel coronavirus) nucleocapsid antibody, mouse mab is mers another sars? investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates contact investigation for imported case of middle east respiratory syndrome seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012. influenza other respir viruses kinetics of serologic responses to mers coronavirus infection in humans hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological findings from a retrospective investigation specific serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study cdc concludes indiana mers patient did not spread virus to illinois business associate middle east respiratory syndrome: what clinicians need to know interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov characteristics of traveler with middle east respiratory syndrome distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms health protection agency ukncit. evidence of person-to-person transmission within a family cluster of novel coronavirus infections prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications respiratory tract samples, viral load and genome fraction yield in patients with middle east respiratory syndrome ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response a case of imported middle east respiratory syndrome coronavirus infection and public health response viral respiratory infections among hajj pilgrims in 2013 influenza a and b viruses but not mers-cov in hajj pilgrims acute respiratory infections in travelers returning from mers-cov-affected areas changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a 10-year retrospective analysis of data soaring mers cases cause pandemic jitters, but causes are unclear co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 recovery from severe novel coronavirus infection human isolate 115. who statement on the fifth meeting of the ihr emergency committee concerning mers-cov | who statement mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia evidence for camel-to-human transmission of mers coronavirus taking stock of the first 133 mers coronavirus cases globally-is the epidemic changing? human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection. zoonoses public health summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in bats, saudi arabia emerging infectious diseases associated with bat viruses bats and their virome: an important source of emerging viruses capable of infecting humans coronavirus diversity, phylogeny and interspecies jumping rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease link between mers virus and camels worries breeders dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) fiqh us-sunnah antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries mers coronavirus neutralizing antibodies in camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus antibody reactors among camels in dubai serologic assessment of possibility for mers-cov infection in equids mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate human infection with mers coronavirus after exposure to infected camels, saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus the holy qur'an sacrificing an animal at mina occupational exposure to dromedaries and risk for mers-cov infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels the effects of temperature and relative humidity on the viability of the sars coronavirus viability of pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? an observational, laboratory-based study of outbreaks of mers-coronavirus in jeddah and riyadh, kingdom of saudi arabia assessment of the mers-cov epidemic situation in the middle east region assessing the pandemic potential of mers-cov interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility transmission dynamics and control of ebola virus disease (evd): a review health care worker contact with mers patient, saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control measures age-specific and sex-specific morbidity and mortality from avian influenza a(h7n9) mers-cov outbreak in jeddah-a link to health care facilities infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers-cov recombination: implications about the reservoir and potential for adaptation middle east respiratory syndrome coronavirus recombination and the evolution of science and public health in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 reliable typing of mers-cov variants with a small genome fragment variations in spike glycoprotein gene of mers-cov molecular epidemiology of hospital outbreak of middle east. respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) in the republic of korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities followup of contacts of middle east respiratory syndrome coronavirus-infected returning travelers, the netherlands press release -who, korea-who joint mission on mers cov call for infection control to stem mers infection control and mers-cov in health workers an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome coronavirus (mers-cov) -republic of korea samsung hospital to invest w100b in post-mers improvements why the panic? south korea's mers response questioned intensified public health measures help control mers-cov outbreak in the republic of korea south korean mers outbreak spotlights lack of research complete genome sequence of middle east respiratory syndrome coronavirus isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china urgent call for research on middle east respiratory syndrome (mers) in korea middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak no respite for korean economy even as mers patients recover middle east respiratory syndrome coronavirus (mers-cov) -china mers in south korea and china: a potential outbreak threat? objective determination of end of mers outbreak, south korea updates on mers outbreak (as of 6:00 on surveillance operation for the 141st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission moh holds the first press conference to update community and media on mers severe acute respiratory syndrome vs. the middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) 2012-2015 case list of moh/who novel coronavirus mers ncov announced cases geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data recent evolution patterns of the middle east respiratory syndrome coronavirus (mers-cov) preliminary analysis of middle east respiratory syndrome coronavirus (mers-cov) sequences from korea and china any unreferenced opinions expressed herein are those of the authors and do not necessarily represent the views of any employer or institution. our thanks go to andrew rambaut, maia majumder, marion koopmans and hale abdali for helpful discussions on social media and cmam and riam for patience. the authors declare that they have no competing interests.author's contributions imm and kea contributed equally to this work and read an approved the final manuscript. key: cord-337825-ujq9mxk7 authors: chen, bin; tian, er-kang; he, bin; tian, lejin; han, ruiying; wang, shuangwen; xiang, qianrong; zhang, shu; el arnaout, toufic; cheng, wei title: overview of lethal human coronaviruses date: 2020-06-10 journal: signal transduct target ther doi: 10.1038/s41392-020-0190-2 sha: doc_id: 337825 cord_uid: ujq9mxk7 coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. seven coronaviruses that infect humans have been identified: hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, sars-cov, mers-cov, and sars-cov-2. among them, sars-cov and mers-cov caused outbreaks in 2002 and 2012, respectively. sars-cov-2 (covid-19) is the most recently discovered. it has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. viruses are genetically simple, yet highly diverse. however, the recent outbreaks of sars-cov and mers-cov, and the ongoing outbreak of sars-cov-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. this paper mainly focuses on sars-cov, mers-cov, and sars-cov-2. here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment. coronaviruses are crown-like particles with spikes protruding from their surface. they are enveloped viruses with single-stranded, positive-sense rna (+ssrna) genomes of approximately 26-32 kb, which is currently the largest known genome size for an rna virus. 1 rna viruses are divided into five branches. 2, 3 certain groups in a particular branch might be related to and/or share features with viruses classified in another branch. in branch 1 are the leviviruses and eukaryotic relatives (e.g., mitoviruses, narnaviruses, ourmiaviruses); branch 2 represents several +rna virus families (of eukaryotes) (e.g., orders picornavirales and nidovirales, and the families caliciviridae, potyviridae, astroviridae, and solemoviridae) and several dsrna viruses (e.g., partitiviruses and picobirnaviruses); in branch 3 are certain +rna viruses such as the supergroups of alphaviruses and offlaviviruses, the nodaviruses, tombusviruses, and many small and novel groups; in branch 4 are dsrna viruses such as the cystoviruses, reoviruses, and totiviruses; branch 5 consists of −rna viruses. 2 coronaviruses were classified as most likely related to branch 2, and belong to the order nidovirales, and the family coronaviridae. in 2018, the international committee on taxonomy of viruses divided the coronaviridae into the orthocoronavirinae and letovirinae subfamilies. according to their host targets, coronaviruses can also be divided into animal and human coronaviruses. many diseases in domestic animals are related to animal coronaviruses, such as canine respiratory coronavirus, which causes respiratory disease in dogs. 4 the highly pathogenic human coronaviruses belong to the subfamily coronavirinae from the family coronaviridae. the viruses in this subfamily group into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the classic subgroup clusters are labeled 1a-1b for the alphacoronaviruses and 2a-2d for the betacoronaviruses. to date, including the recently discovered sars-cov-2, there are seven coronaviruses that infect humans. human coronavirus (hcov)-229e, hcov-nl63, hcov-oc43, or hcov-hku1 cause only the common cold, 5 whereas the severe acute respiratory syndrome coronavirus (sars-cov) or the middle east respiratory syndrome coronavirus (mers-cov) cause relatively high mortality and emerged in 2002 6 and 2012, 7 respectively. notably, sars-cov-2 is currently causing a worldwide epidemic. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, 1, 8 whereas sars-cov-2 is a new member of the betacoronaviruses distinct from sars-cov and mers-cov. 9 figure 1 shows the phylogenetic tree of rna viruses. the estimated mutation rates of coronaviruses (covs) are moderate to high compared to those of other ssrna viruses. 1 there are two gene loci that are sites of variation in sars-cov. one of these sites is located in the spike (s) protein gene. the second major site of variation is the accessory gene open reading frame orf8. in mers-cov, the major sites of variation are located in the s, orf4b, and orf3 genes. 2 the major differences in the sequence of the s gene of sars-cov-2 are three short insertions in the n-terminal domain and changes in four out of five of the key residues in the receptor-binding motif. 9 the organizations of the genome and gene expression are similar for all coronaviruses. orf1a/b, located at the 5′ end, encodes 16 nonstructural proteins (nsps) (named nsp1-nsp16); other orfs at the 3′ end encode structural proteins, including the s, envelope (e), membrane (m), and nucleocapsid (n) proteins. the mutations in sars-cov-2 have become a hot research topic. the surface proteins of sars-cov and batcov-ratg13, the nearest potential bat precursors of sars-cov-2, have 76 and 97% sequence identity, respectively, to that of sars-cov-2. 10 compared to that of sars-cov, the antigenic surface of sars-cov-2 is highly divergent compared to other covs. since its outbreak began at the end of 2019, sars-cov-2 has acquired mutations throughout its genome, and there are already hundreds of virus strains distributed worldwide. according to gisaid.org, as of may 8, there were 16,004 full genome sequences available, and the s clade of the full genome tree contains the largest group of viruses, indicating that mutations in orf8-l84s are most frequent. the temporal resolution assumes an average nucleotide substitution rate of 5 × 10 −4 substitutions per site per year. based on these mutations, researchers found that sars-cov-2 can be divided into two subtypes. they reported that the snps at locations 8782 and 28,144 show strong linkage; the "ct" haplotype was defined as the "l" type because t28,144 encodes leucine, while the "tc" haplotype was defined as the "s" type because c28,144 encodes serine, 11 which is in accordance with another study that divided sars-cov-2 into two types. 12 the "l" type seems to be more prevalent and aggressive, but the "s" type may be ancestral, as sites 8782 and 28,144 were identical to the orthologous sites in the most closely related viruses. 13 patients may be infected by either or both types. 11 from the gisaid update, compared with ratg13, there are differences in the receptor-binding interface, which may have favored host switching. data related to the sequence and mutations of sars-cov-2 are also available and continuously updated online through the china national center for bioinformation (cncb) (https://bigd.big.ac.cn/ncov). the history of sars-cov, mers-cov, and sars-cov-2 the first case of sars was discovered in foshan, china, in november 2002. 6 infections occurred through either direct or indirect contact with patients. by july 2003, sars-cov had spread to over 30 countries, 12 causing 8096 reported cases and 774 deaths. after that, no additional infections were detected, and the sars pandemic was declared over. sars-cov was first isolated from himalayan palm civets (hpcs) from a live-animal market in guangdong, china. 12, 14 other animals, such as raccoon dogs (nyctereutes procyonoides), along with human workers from the same market, also showed evidence of viral infection. 12 multiple studies have demonstrated that the reservoirs of several coronaviruses, including sars-cov-like and mers-cov-like viruses, were bats. 8, 15 although palm civets might have been intermediary hosts of sars-cov, 16 researchers often concluded there was no evidence that these animals were the ultimate sources of sars-cov, and the viruses cannot circulate directly in palm civets in the wild. 17 in 2003, guan and zheng's team investigated a live-animal retail market in guangdong, focusing on recently captured wild animals and human consumption. the animals sampled included seven wild and one domestic animal species. they collected nasal fig. 1 phylogenetic tree of coronaviruses based on full-length genome sequences. all complete genome sequences of coronavirus were downloaded from the ncbi reference sequence database, refseq. the tree was constructed using maximum likelihood estimation (mle) by mega x, with clustal omega as the multiple sequence alignment method, and 1000 bootstrap replicates. only bootstraps ≥50% values are shown. the seven known human-infecting coronaviruses are indicated with a red star and fecal samples with swabs and then used reverse transcriptionpolymerase chain reaction (rt-pcr) to test for viral nucleic acid from the n gene of the human sars-cov. the rt-pcr assay results showed that samples from four of six hpcs were positive; the other seven species (beaver, chinese ferret-badger, chinese hare, chinese muntjac, domestic cat, hog-badger, and raccoon dog) sampled were negative. 12 mers-cov was first found in 2012 in a lung sample from a 60year-old patient who died of respiratory failure in jeddah, saudi arabia. on 15 september 2012, a similar type of virus named human coronavirus was isolated from a patient with severe respiratory infection. cases have also been reported in other countries. 18 mers has a 35% mortality rate, and since it emerged in the human population in june 2012, it has caused substantial morbidity and mortality. 19, 20 from 2012 until january 15, 2020, the total number of laboratory-confirmed mers-cov infection cases reported globally to the world health organization (who) was 2506, with 862 associated deaths, covering 27 countries (www. who.int/csr/don/31-january-2020-mers-united-arab-emirates/en). cases of mers from other countries were linked to travel to the middle east. 20,21 mers-cov was identified from the saliva of a patient with acute pneumonia and renal failure in jeddah (ksa). mers-cov can be detected in respiratory tract secretions, as well as in feces, serum, and urine. [22] [23] [24] in addition, it has been isolated from environmental objects such as bedsheets, bedrails, intravenous fluid hangers, and x-ray devices. 25 in the case of known camel infection, mers-cov was transmitted from a camel to a human, which was confirmed by rna sequencing. 26 at the end of december 2019, the first clusters of patients with pneumonia caused by sars-cov-2 were reported in wuhan, china. 27 the basic reproduction number of sars-cov-2 is approximately 2.2, indicating that each patient would on average spread the infection to 2.2 people. 28 human-to-human transmission of sars-cov-2 occurred rapidly, and the atypical symptoms during the early stage may be a further disadvantage. 29 moreover, human infection might have begun months prior to the official announcement of the outbreak. 30 to prevent further spread, stricter screening and surveillance are needed at travel hubs. 31 early detection, diagnosis, and treatment are also effective measures to contain the epidemic. 32 nonetheless, the fatality rate of sars-cov-2 is lower than that of sars, 33 with an estimated mortality risk of 2%. 34 sars-cov-2 was first isolated from clinical specimens using human airway epithelial cells and the vero e6 and huh-7 cell lines. 27 approaches to assess virions include isolation from lower respiratory tract specimens 35 and artificially infected specific pathogen-free human airway epithelial cells. 36 fluorescence quantitative pcr with primers designed according to specific sequences and serological testing aimed at igg and igm can be used to detect the virus. 9 sars-cov, mers-cov, and sars-cov-2 pose major challenges to global health, causing infections in large parts of the world. sars-cov was found in 30 countries and mers-cov in 27 countries, while sars-cov-2 was found in 213 countries by 8th may, 2020. details on the pandemics caused by sars-cov-2 are shown in box 1, highlighting the status of the coronavirus-caused diseases worldwide. epidemiological analysis and symptoms of sars, mers, and coronavirus disease 2019 (covid19) the clinical manifestations of sars include hypoxia, cyanosis, high fever (above 38°c), accelerated breathing or respiratory distress syndrome, and shortness of breath. x-rays show changes to the lungs to varying degrees. the who case definition (2003) includes the following: (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive test for sars-cov by one or more assays. 37 similar to sars-cov infection, mers-cov infection manifests as a severe lower respiratory tract infection with extrapulmonary involvement and high fatality rates. 19 symptomatic patients may present fever, chills, stiffness, myalgia, discomfort, cough, shortness of breath, and gastrointestinal symptoms of diarrhea, vomiting, and abdominal pain. pneumonia is common, and severe infection with acute respiratory failure, renal failure, and shock is particularly frequent among older patients. 20 previous studies have estimated that approximately 12.5-25% of mers-cov infections may be asymptomatic. 20, 38 it must be noted that immunocompromised people are at high risk of mers-cov infection. 21 for covid-19, fever, cough, myalgia, and fatigue are most commonly observed at illness onset; less commonly observed are sputum production, hemoptysis, and headache, among others. 29, 35 similar to sars and mers, some patients develop acute respiratory distress syndrome (ards). 39 around 81% of infected patients only develop mild symptoms, with mild pneumonia or no pneumonia. 14 and 5% are severe and critical status, respectively. 40 patients requiring icu care tend to be much older and are more likely to have underlying comorbidities, such as hypertension and diabetes. 29 additionally, symptoms such as pharyngeal pain and anorexia are reported at higher rates among those admitted to the icu than among non-icu patients. 29 histopathologically, symptoms such as diffuse alveolar damage (dad), loss of cilia, squamous metaplasia, denudation of bronchial epithelia, and giant-cell infiltrate often occur in the lungs of patients with sars. the number of macrophages increases prominently in the alveoli and the interstitium. 41 according to the morphological changes, observed, most authors have subclassified lung lesions in sars into two or three consecutive phases: an acute exudative inflammatory phase, a fibrous proliferative phase, and a final fibrotic stage, although there is considerable overlap in histological findings among these phases. 42 the main features of the first phase include extensive hyaline membrane formation, edema, alveolar hemorrhage, and fibrin exudation in alveolar spaces. the second phase includes widening of septae, pneumocyte hyperplasia, and organizing fibromyxoid and cellular exudates. 42, 43 the third phase includes septal and alveolar fibrosis. 42 several autopsies of sars patients have demonstrated secondary bronchopneumonia, and the pathogens identified mainly consist of staphylococcus aureus, mucor sp., aspergillus sp., pseudomonas aeruginosa, and cytomegalovirus. 43 sars-cov also greatly affects the immune system. for example, hemorrhagic necrosis is usually obvious in the lymph nodes and spleen. 44 as described above, sars-cov attacks immune cells, especially t cells and macrophages, with approximately 50% of lymphocytes and 30% of monocytes in the circulation becoming infected. 42, 45 under the influence of the virus, the patient's germinal centers disappear, and both t and b lymphocytes are depleted. in the spleen, the white pulp shrinks, hemorrhage and necrosis occur in the red pulp, and the periarterial lymphatic sheaths decrease. 42, 45, 46 with regard to the immune system, sars-cov can infect the lymphoid component of the intestine. for this reason, the patient's submucosal lymphoid tissue atrophies. 47 sars-cov can also infect epithelial cells in the mucosa of the small and large intestines, and the digestive tract may undergo mild diffuse inflammation and atrophy of the submucosal lymphoid tissues. 47 according to previous reports, symptoms in the liver include extensive hepatocyte mitosis, balloon degeneration of hepatocytes, mild to moderate lymphocytic infiltrates, fatty degeneration, and central lobular necrosis. 42 additionally, apoptosis of liver cells has been confirmed in some cases. 48 on autopsy, the kidneys of some sars patients showed focal bleeding and acute tubular necrosis of different degrees. one of the major complications of ards is acute kidney injury. 42, 49, 50 in many other cases, the features are nonspecific, including benign hypertensive nephrosclerosis and autolysis. 42 moreover, researchers have detected viral particles and genome sequences in the cytoplasm of neurons of the hypothalamus and cortex in the brain, 51 which suggests that the virus can cross the blood-brain barrier and cause degeneration and necrosis of neurons, edema, extensive glial cell hyperplasia, and cellular infiltration. 42 pneumocytes and epithelial syncytial cells are important targets of mers-cov. the lung tissue presents dad. severe peripheral vascular diseases, patchy cardiac fibrosis, and hepatic steatosis have also been found in other organs. 52 another symptom is bronchial submucosal gland necrosis. 52 renal biopsy may show acute tubulointerstitial nephritis and acute tubulosclerosis with protein casts. 22 in covid-19 patients, a bilateral (sometimes unilateral) distribution of patchy shadows and ground glass opacity in the lungs is typical, based on ct scans. 29 plasma concentrations of interleukins (ils) 2, 7, and 10, granulocyte-colony stimulating factor, interferon (ifn)-γ-inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and tumor necrosis factor α are increased in most dying patients. 39, 53 in addition, neutrophilia related to cytokine storms induced by virus invasion, coagulation activation related to a sustained inflammatory response, and acute kidney injury related to the effects of the virus, hypoxia, and shock, may be involved in mortality. 29 those who survive intensive care may experience long-term lung damage and fibrosis due to aberrant and excessive immune responses. 39 moreover, researchers found that as sars-cov-2 spread and changed across generations, clinical symptoms started to differentiate those infected previously from those infected more recently. the initial symptoms are becoming more insidious, which indicates that the virus may gradually evolve into an influenza-like virus and lie dormant for longer in asymptomatic patients. 54 in conclusion, in terms of outbreak times, sars-cov was the earliest, followed by mers-cov, then sars-cov-2. to date, mers-cov has the longest duration of infection, and sars-cov-2 appears to be the most infectious. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. the common transmission methods and symptoms of the three coronavirus infection are shown in fig. 2 . structural studies of sars-cov, mers-cov, and sars-cov-2 sars-cov is a coronavirus with a diameter of approximately 100 nm. coronaviruses are the largest +ssrna viruses and contain at least 14 orfs, 16 protein combines with viral rna to form a nucleocapsid, which is involved in the replication of sars-cov and is the most abundant protein in virus-infected cells. 56, 57 the m protein is a membrane glycoprotein that is involved in budding and envelope formation. the s protein of sars-cov is a type i transmembrane (tm) glycoprotein that is responsible for viral binding, fusion and entry into cells, as well as antibody induction. 16, 58 the s protein contains a signal peptide (sp: amino acids 1-12) and three domains called the extracellular domain (amino acids 13-1193), the tm domain (amino acids 1194-1215) and the intracellular region (amino acids 1216-1255). 16 the extracellular domain consists of two subunits: s1 and s2. the fragment located in the middle region of the s1 subunit (amino acids 318-510) is the angiotensin-converting enzyme 2 (ace2) receptor-binding region. the s2 subunit comprises a fusion peptide (fp) and two x 7-valent element repeats (hr1 and hr2) that are responsible for fusion between the virus and the target cell membrane. 58 thus, the s protein may be key to develop a vaccine to generate antibodies and block virus binding and fusion in the coronaviruses (fig. 3) . the 3′ end of the sars virus genome encodes 12 structural proteins and helper proteins, of which orf3a, orf6, orf7a, and orf7b have been proven to be viral structural proteins involved in the formation of viral particles. the 5′ end of the genome encodes 16 nonstructural proteins (nsps), which are important for virus assembly, and may enable the design of small molecule drugs and/or vaccines. mers-cov belongs to lineage c of the genus betacoronavirus (βcov) in the family coronaviridae, order nidovirales, and it is the first lineage c cov and the sixth cov known to cause human infection. mers-cov is an enveloped +ssrna virus similar to other covs, but the amino acid sequence homology between mers-cov and other known covs is less than 90%. 19 orf1a and orf1b located in the first two-thirds of the mers-cov genome are involved in virulence and encode nsps (nsp1-16). the remaining third of the genome encodes four structural proteins, the s, e, m, and n proteins, as well as five accessory proteins (orf3, orf4a, orf4b, orf5, and orf8b). [59] [60] [61] the flanking regions of the genome contain the 5′ and 3′ untranslated regions (utrs). 62 most mers-cov proteins are found in the cytoplasm. 63 to date, the ns4b protein and the orf3a-encoded protein are the only known coronavirus proteins to be detected in the nucleus. 64 nsp1 suppresses host gene expression in infected cells, and its rna cleavage-inducing function promotes virus assembly/budding. 65 nsp16 is a viral 2′-o-methyltransferase (2′-o-mtase) that encodes critical functions in immune modulation and infection, suggesting that nsp16 is a conserved universal candidate for rapid design of a live attenuated vaccine. 66 orf4a's protein can mask viral dsrna to play a role in immune evasion, including type 1 ifn and protein kinase r-mediated antiviral stress responses. 67 mers-cov nsp13 is a full-length coronavirus helicase that contains multiple domains, including an n-terminal cys/his rich domain with three zinc atoms, a beta-barrel domain and a c-terminal superfamily (sf) 1 helicase (sf1) core with two reca-like subdomains. this protein might interfere with the nonsensemediated mrna decay pathway to avoid degradation of viral rna. 68 the mers-cov s protein can recognize dipeptidyl peptidase 4 (dpp4) on the cell surface, promoting the fusion of the viral envelope and the cell membrane. the mers-cov s protein, a 1353-amino acid type i tm glycoprotein located on the viral envelope surface, is composed of s1 and s2 subunits. 69 the structure of the s protein consists of four parts: an sp located at the n terminus (amino acids 1-12), an extracellular domain (amino acids 13-1195), a tm domain (amino acids 1196-1215), and an intracellular domain (amino acids 1216-1255). 16 the s1 subunit contains two independent domains, an n-terminal domain (ntd, amino acids 18-353) 70 and a c-terminal domain (amino acids 367-588), both of which may function as receptor-binding domains (rbds); 71 these domains serve as critical targets for the development of vaccines and therapeutics 72, 73 and facilitate the involvement of the s1 subunit in cell receptor (dpp4) binding. the core structure of the mers-cov s protein rbd is a five-stranded antiparallel sheet with several short helices, in which three disulfide bonds stabilize the core by connecting c383 to c407, c425 to c478, and c437 to c585. 16, 73 the accessory subdomain of the c-domain is a hypervariable region for receptor recognition fig. 3 clustering of the spike protein of each coronavirus. fifty-four spike protein sequences filtered from each coronavirus coding sequences were clustered using the clans (cluster analysis of sequences) program on the website of mpi bioinformatics toolkit. each colored dot represents the spike protein sequence of each coronavirus. dots in the same color mean they are of the same genus, and each line shows the similarity of two sequences, with darker lines indicating higher similarity (lower e values). the coronaviridae family includes the following genera: alphacoronavirus (colored in green); betacoronavirus (red); gammacoronavirus (orange), and deltacoronavirus (blue). the indicated sars-cov, mers-cov, and sars-cov-2 belong to the genus of betacoronavirus and is called the receptor-binding motif (rbm). the accessory subdomains in sars-cov and mers-cov differ, resulting from divergent evolution and leading to the use of different receptors. 71 dpp4 consists of an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets 74 and the rbd. the s2 subunit of mers-cov contains an fp (residues 943-982), an hr1 domain (residues 984-1104), an hr2 domain (residues 1246-1295), a tm domain (residues 1296-1317), and an intracellular domain (residues 1318-1353). 69 the s2 subunit is responsible for fusion between the virus and the target cell membrane. after the s1 subunit binds to dpp4, the s2 subunit inserts its fp into the target cell membrane to change its conformation. its hr1 helix then forms a homotrimer with an exposed surface that has three highly conserved hydrophobic grooves, and binds with hr2 to form a six-helix bundle (6-hb) core structure, which facilitates fusion by bringing the viral and cell membranes into close proximity. 69 the genetic material of mers-cov then enters the host cell via the fusion pore. 69 sars-cov-2, which causes covid-19, is a spherical betacoronavirus with a diameter of 60-140 nm and unique spikes ranging from 9 to 12 nm. 27 its rna has the typical betacoronavirus organization, containing a 5′ utr, a gene encoding the replicase complex (orf1a/b), the s gene, e gene, m gene, and n gene, a 3′ utr, and several unidentified nonstructural orfs. 27, 75 the length of the sequences is 265 nt for the 5′ terminal region, 229 nt for the 3′ terminal region, 3822 nt for the s gene, 228 nt for the e gene, 669 nt for the m gene, and 1260 nt for the n gene. 75 the 21,291nt-long orf1a/b gene contains 16 predicted nsps. 75 similar to sars-cov, there is a predicted orf8 gene that is 366 nt in length and located between the m and n orf genes. 75 nucleotides 1, 1029, and 1652 may be the recombination breakpoints because based on the fragments from nt 1 to nt 1029 and nt 1652 to the end, sars-cov-2 seems to be related to bat-sl-covzc45 and bat-sl-covzxc21; however, when considering the region from nt 1030 to nt 1651, sars-cov-2 is mostly likely to be grouped with sars-cov and bat sars-like covs. more research is required to analyze the recombination of the genome as a whole. 75 with over 10% of its conserved replicase complex being different from those of other betacoronaviruses, sars-cov-2 solely occupies a newly created subclade within the sarbecovirus subgenus. 27, 75 bats may be its natural host, but owing to several arguments including the complicated environment in the wet markets in wuhan, this remains unclear, and further research is needed. 36, [75] [76] [77] the free energy of the spike protein of sars-cov-2 is much lower than that of sars-cov, indicating that sars-cov-2 is more stable than sars-cov. 78 the high similarity of the rbd sequences and of the spike protein structures between sars-cov-2 and sars-cov, 36, 75, 79 also the simulation of the spike protein of sars-cov-2 binding to ace2, 80 the receptor of sars-cov, and results shown that ace2 plays a vital role in sars-cov-2 entry into hela cells, 76 indicating that sars-cov-2 also uses ace2 for cell entry. structural modeling of the ace2-b0at1 complex (b0at1 is used to obtain stable ace2) suggests that the complex can bind two s proteins simultaneously, providing important clues to the molecular basis of coronavirus recognition and infection. 81 the rbd-ace2 binding free energy for sars-cov-2 is significantly lower than that for sars-cov, in accordance with the fact that sars-cov-2 is more infectious than sars-cov. 78 a recent study found that cd147, a kind of tm glycoprotein, facilitates cell entry of sars-cov-2 functionally, and its affinity constant with the s protein is 1.85 × 10 −7 m. 82 table 1 shows a comparison of the structures of sars-cov, mers-cov, and sars-cov-2. the specific function of the sars-cov-2 proteins, including s, e, m, and n proteins, need further study. in conclusion, sars-cov, mers-cov, and sars-cov-2 are similar in structure. their major proteins are structural proteins, including the s, e, m, and n proteins, accessory protein, and nsps (nsp1-16). all these viruses rely on the s protein to identify cell targets and fuse with membranes. their s proteins are composed of s1 and s2 subunits. the s1 subunit contains an ntd and a c-domain, in which there is an rbd. the accessory subdomain of the c-domain is a hypervariable region for receptor recognition and is called the rbm. the difference is that sars-cov recognizes ace2, sars-cov-2 recognizes ace2 and cd147, but mers recognizes dpp4, which results from the difference between the accessory subdomains of sars-cov, sars-cov-2, and mers-cov. the function of the accessory and nonstructural proteins of these three viruses is related to viral replication and assembly, hence linked to virulence. pathogenesis of sars-cov, mers-cov, and sars-cov-2 sars-cov and sars-cov-2 are nearly identical in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4. the human ace2 protein is a typical zinc metallopeptidase that comprises 805 amino acids and contains a single catalytic domain. it is a type i integral membrane glycoprotein that is oriented with an extracellular n terminus and a catalytic site that functions in metabolizing circulating peptides. 83 it is believed that there is a virus-binding hot spot on ace2 that is not pre-existent or preorganized but is induced to form upon virus binding. 84 the hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. the general structural features of the hot spot favor virus binding: it is located in a region on ace2 that is furthest from the membrane, relatively flat, and free of glycosylation and is thereby easily accessible to viruses. the hot spot is mainly an intrinsic property of ace2, but it is also a dynamic structure and receives structural contributions from both ace2 and the viruses, with the contributions of ace2 being more pronounced. dpp4 is a type ii transmembrane glycoprotein that consists of approximately 766 amino acids. dpp4 contains an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets. 74 the s protein rbd binds to the side surface of the dpp4 propeller and the amino acid residues in lancets 4 and 5, including k267, q286, t288, r317, r336, q344, a291, l294, and i295. 74 the pathogenic mechanism of sars-cov is believed to include two parts: the virus injures target cells directly, and subsequent immune system dysfunction leads to indirect injury. 42 sars-cov spreads via droplet and contact transmission and via the fecal-oral route. through droplet inhalation, the virus enters the respiratory tract and invades epithelial cells. infection and replication of the virus and local inflammatory changes contribute to lung tissue damage. subsequently, sars-cov infects circulating and resident immune cells. the key cells in this step consist mainly of t cells and macrophages. the viruses are then carried to other organs, including secondary lymphoid organs, by these circulating immune cells. 42 sars-cov resembles hiv because both viruses attack immune cells and cause immunodeficiency. 42 sars-cov causes lung injury mainly by inhibiting the function of ace2. the virus binds to ace2 via the spike protein, downregulating its function and contributing to lung injury. ace2 is an important component of the renin-angiotensin system (ras). in this system, angiotensinogen is first converted to angiotensini (angi) by renin, and then angiis converted to ang ii under the influence of ace2. ace2 downregulates the levels of angi and ang ii, which can bind to the ang ii type i (at1) receptor and cause certain types of lung injury, mainly pulmonary hypertension, pulmonary fibrosis, and acute lung injury. 85 ang ii can directly induce pulmonary artery smooth muscle cells to grow or proliferate rapidly through at1, thus causing pulmonary hypertension. 86 ang ii contributes to pulmonary fibrosis by promoting the expression of the profibrotic cytokine transforming growth factor-b1, causing fibroblasts to convert to myofibroblasts and accumulate collagen. 70 several strategies can be used by the virus to escape the innate immune response. normally, viral pathogen-associated molecular patterns (pamps) are detected by host pattern recognition receptors (prrs). pamps include viral dsrna and mrna, and prrs include retinoic acid-inducible gene i protein (rig-i) and melanoma differentiation-associated protein 5 (mda5). production of type i ifn is induced via the nuclear factor-κb (nf-κb) pathway. when ifn binds to the ifnα/β receptor, signal transducer and activator of transcription (stat) proteins are activated, which increases the production of other antiviral proteins and thus blocks sars-cov replication for further infection. 11 additionally, sars-cov could encode several proteins that hinder the signaling cascades downstream of prrs. the cell receptor of mers-cov is dpp4, which is widely expressed on epithelial cells in the prostate, alveoli, kidneys, liver, and small intestine and on activated leukocytes; thus, the range of mers-cov tissue tropism is broader than that of any other similar coronavirus. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of proinflammatory cytokines and chemokines (such as tnf-α, il-6, cxcl-10, ccl-2, ccl-3, ccl-5, and il-8). 87 compared to sars-cov infection, mers-cov infection can cause higher expression levels of il-12, ifn-γ, ip-10/cxcl-10, mcp-1/ccl-2, mip-1α/ccl-3, rantes/ccl-5, and il-8. 87 induction of immune cell-recruiting cytokines/chemokines might lead to the infiltration of a large number of immune cells into the lower respiratory tract, causing severe inflammation and tissue damage. 87 mers-cov can also infect t cells and lead to apoptosis, avoiding the host immune response and facilitating faster spread. 88 mers-cov has also evolved a mechanism to escape the host cell immune system. when host sensors initiate signaling pathways, transcription of type i ifn genes begins, and the type i ifn response, which is an essential component of antiviral innate immunity, is initiated. [89] [90] [91] ifn activates the transcription of numerous ifn-stimulated genes (isgs) and synthesizes 2′,5′oligoadenylate (2-5a), which causes rnase l activation. [91] [92] [93] activated rnase l can cleave both viral and host ssrna, leading to translation stagnation and apoptosis and limiting virus replication and transmission in vitro and in vivo. 92, 94 however, the mers-cov protein ns4b has phosphodiesterase activity and antagonizes rnase l via enzymatic degradation, leading to inference with ifn signaling. although ns4b is mainly expressed in the nucleus, the expression level of the ns4b protein in the cytoplasm is sufficient to prevent activation of rnase l. 88 in addition, the mers-cov m, orf4a, orf4b, and orf5 proteins are reported to be strong ifn antagonists, among which the orf4a, orf4b, and orf5 proteins inhibit both type i ifn induction 61, 95, 96 and nf-κb signaling pathways, which are similar to the type i ifn signaling pathway, 96 providing a possible mechanism for mers-cov evasion of innate immunity. 88, 96 for sars-cov-2, the first cluster of patients was believed to be associated with a seafood market. 27 however, due to the identification of patients without direct exposure to the market, transmission between humans was proven. 97 hospital-related transmission has also been detected via infected medical workers and hospitalized patients. main routes of transmission include respiratory droplets, close contact, and extended exposure to aerosol environments loaded with high concentrations of virions. in addition to transmission through the respiratory tract, exposure of unprotected eyes to sars-cov-2 might cause acute respiratory infection. 98 notably, the virus can be transmitted during the incubation period. 99 the tm protease serine type 2 (tmprss2) primes the s protein of sars-cov-2 for cell entry. 100 based on analysis of the ace2 rna expression profile in normal human lungs, sars-cov-2 mainly infects type ii alveolar (at2) cells (only 0.64% of human lung cells can express ace2, 83% of which are at2 cells), and they express many other genes that facilitate sars-cov-2 infection; 101 moreover, cd147 was also reported that it probably functionally facilitates cell entry of sars-cov-2. 82 high expression of ace2 has also been detected in cells of the digestive system, such as upper esophageal cells; accordingly, this system is a potential route of infection. 102 in addition to the lungs and intestines, other organs, such as the heart, esophagus, kidney, bladder, testis, ileum, and adipose tissue, also express ace2, and the expression level is higher than that in the lungs. 103, 104 certain tumor tissues have higher expression of ace2, making cancer patients more vulnerable than other people. 104 in conclusion, sars-cov and sars-cov-2 bind cells expressing ace2. they affect the ras system due to the affinity for ace2 (much stronger with sars-cov-2 than with sars-cov), leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. the specific pathogenic mechanisms of these three viruses are illustrated in fig. 4 . diagnostic methods in addition to clinical manifestations, definitive diagnosis and confirmation of a coronavirus infection requires specific testing. the criteria for the diagnosis of sars from the who include (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive tests for by at least one assay. as for mers, the criteria for the diagnosis according to the who include (1) fever, cough, and hospitalization with suspicion of lower respiratory tract lesion, (2) history of contact with probable or confirmed cases, (3) history of travel or residence within the arabian peninsula, and (4) positive tests by a pcr-based detection method using respiratory samples, such as nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal) and induced sputum, or by serological tests such as the rapid immunochromatographic assay using highly selective monoclonal antibodies at room temperature. the initial criteria for the diagnosis of sars included (1) a history of travel or residence in wuhan within 2 weeks before the onset of illness, (2) exposure to patients from wuhan with fever and respiratory symptoms within 2 weeks before the onset of illness or the existence of clusters of diseases, (3) fever accompanied by radiographic features of pneumonia, a normal or decreased total number of white blood cells, or a decreased lymphocyte count, and (4) fluorescence pcr testing positive for nucleic acids of sars-cov-2. 37, 59, [105] [106] [107] early diagnosis of mers is difficult, but several highly specific and sensitive molecular and serologic assays exist for diagnosis in both animals and humans. 108 among them, a rapid immunochromatographic assay can detect mers-cov antigens based on the detection of the mers-cov nucleocapsid protein in a short time frame; the relative sensitivity and specificity of this test are 93.9 and 100%, respectively. 105 nucleic acid tests, such as real-time pcr tests, were mostly used to detect virus in the early stage of the outbreak of sars-cov-2, although this approach requires a relatively long time and is not conducive to accelerating diagnosis or large-scale application. therefore, alternative, rapid diagnostic kits for sars-cov-2 may be used. for example, the colloidal gold detection kit uses serum, plasma, or whole blood samples to detect igm/igg antibodies for diagnosis on the 7th day of infection or the 3rd day after onset and requires only 15 min. the nucleic acid detection kit for six respiratory viruses, including sars-cov-2 and influenza a virus, uses thermostatic amplifier chips for diagnosis. by detecting different infections, it may help distinguish people with influenza from those with other viral infections. a newly developed fig. 4 pathogenesis of sars-cov, mers-cov, and sars-cov-2. sars-cov and sars-cov-2 play a pathogenic role by inhibiting ace2. under the influence of renin and ace, angiotensinogen is converted into ang ii. through the at1 receptor, ang ii acts as a lung injury-promoting factor, and in some cases, may cause vascular constriction, an inflammatory response, cell proliferation, fibrosis, and apoptosis of alveolar epithelial cells, resulting in diseases such as pulmonary hypertension, pulmonary fibrosis, and acute lung injury. ace2 converts ang ii into ang (1-7), and through the mas receptor, ang (1-7) play roles in vasodilation, antiproliferative activity, and antioxidant activity. sars-cov downregulates the activity of ace2 and causes an increase in the amount of ang ii and lung injury. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of pro-inflammatory cytokines and chemokines, leading to a large number of immune cells infiltrating a patient's lower respiratory tract, causing severe inflammation and tissue damage. mers-cov can infect t-cells from human lymphoid organs and causes the peripheral blood inducing apoptosis by intrinsic and extrinsic pathways, thus avoiding host immune response detection method, nanopore targeted sequencing, also has the potential for efficiently detecting viruses in a reasonable time. moreover, it could identify 40 other respiratory viruses and monitor changes in virulence and transmissibility caused by viral mutations. 109 therapeutic agents all patients should be treated in isolation. 53 based on previous studies, drugs may be developed to inhibit cell entry. several strategies have been investigated to identify specific antivirals targeting the s protein, including mers-rbd-targeted nabs that block viral attachment, peptide inhibitors targeting s2 to prevent the formation of the fusion core, and small molecules without defined mechanisms. 110 sdpp4 has been found to bind mers-cov rbd with high affinity, suggesting that sdpp4 could serve as a blocker for mers-cov attachment to and entry into cells. 111, 112 one study reported a monoclonal antibody, 7d10, that binds to the ntd to inhibit cellular entry of mers-cov. 113 experiments have shown that 7d10 not only inhibits the binding of mers-cov to cells but also has effects after viral-cell attachment. 7d10 inhibits virus invasion by blocking the binding of the s1 subunit to the receptor and interfering with the conformational transformation of the s2 subunit when the virus fuses with the cell membrane. 113 a peptide fragment spanning residues 736-761 of the s protein exerts neutralization activity by inhibiting membrane fusion and the entry of mers-cov, suggesting that it is possible to develop vaccines targeting this neutralizing epitope. 114 griffithsin, a lectin derived from red algae, binds to oligosaccharides on the surface of sars-cov s protein and may also prove effective against sars-cov-2. 34, 115 researchers found that antibodies raised against sars-cov, such as css-2, -3, -4, and -5, could cross-neutralize sars-cov-2 by reducing s-driven cell entry, although the efficiency was lower than that observed for sars-cov. 100 meplazumab, an anti-cd147 humanized antibody, binds with the cd147 in competition with the s protein, 116 thus significantly inhibiting virus from invading cells and reducing the time of negative conversion. 82, 106 chloroquine could effectively inhibit sars-cov-2 in vitro, and hydroxychloroquine was even more potent than chloroquine. 117, 118 hydroxychloroquine could significantly help reduce the virus load, and azithromycin could reinforce its effect. 119 arbidol could also contribute to condition improvement. 120 note that these studies were generally limited and further trials and development are necessary. protease inhibitors also have the potential for coronavirus therapeutics. protease inhibitors, including disulfiram, lopinavir, and ritonavir, have been reported to be effective against sars and mers, and disulfiram, a drug for alcohol dependence, was reported to inhibit the papain-like protease (plpro) of mers-cov and sars-cov in cell cultures. 34, 108 in vitro, sars-cov can be inhibited by both 6-mercaptopurine and 6-thioguanine targeting plpro. 121 when used together with ribavirin, the protease inhibitors lopinavir and ritonavir have improved the outcomes of patients with sars compared with those achieved with the use of ribavirin alone. 122 such inhibitors are also being tested in patients infected with sars-cov-2, but whether these inhibitors effectively suppress 3clpro and plpro of sars-cov-2 remains controversial. 34 drugs such as colistin, valrubicin, icatibant, bepotastine, epirubicin, epoprostenol, vapreotide, aprepitant, caspofungin, perphenazine, prulifloxacin, bictegravir, nelfinavir, and tegobuvir bind to the main protease of sars-cov-2, and thus, are potential candidates. 123 lianhua-qingwen formula, a kind of traditional chinese medicine, has been also recently described theoretically as a potential treatment candidate against the main virus protease. 124 the serine protease inhibitor camostat mesylate could partly block sars-cov-2 infection of lung cells by inhibiting tmprss2. 100 viral rna synthesis blockers also have potential. remdesivir has broad-spectrum activity against mers-cov and sars-cov in cell cultures and animal models and is currently in clinical development for the treatment of ebola virus disease. 34, 125 it was also reported positively based on a patient with sars-cov-2 infection in the us, 126 and another study showed that it was able to inhibit the virus. 117 ribavirin is often used to treat sars and mers, but it is uncertain whether it is potent enough against covid-19. 34, 108 regardless, when used alone, ribavirin has minimal activity against sars-cov and may cause strong side effects. it is often used together with corticosteroids. 14,127 a preclinical study of galidesivir (bcx4430), an adenosine analog, revealed antiviral activity against sars-cov and mers-covcov. 115 a study of favipiravir (t-705), a guanine analog, proved its activity against sars-cov-2, and randomized trials of favipiravir plus interferon-α (chictr2000029600), and favipiravir plus baloxavir marboxil (chictr2000029544) are in progress. 34 corticosteroids may be used to treat sars patients to suppress lung inflammation; although this therapy is not associated with mortality in patients with mers, it is associated with delayed clearance of mers-cov rna. 14,128 moreover, clinical evidence does not support the use of corticosteroids for covid-19 treatment. 129 regarding antibodies, plasma therapy using convalescent plasma from fully recovered patients is effective against these coronaviruses, including sars-cov-2. 108, 130 tocilizumab (antihuman il-6 receptor) may curb immunopathology and trials are ongoing now. 131 a study of novel monoclonal antibodies against the mers-cov spike protein suggests that mabs can be utilized for the identification of specific mutations of mers-cov. 132 wang et al. provided an all-hydrocarbon-stapled peptide that likely mimics the native conformation of the c-terminal short α-helical region of the mers-cov s protein, which can block the formation of the hexameric coiled-coil fusion complex to inhibit viral-cell membrane fusion. 133, 134 current treatments for sars mainly include ribavirin, ifn α, plasma therapy, host-directed therapies, and systemic corticosteroids. 14, 41 due to the lack of clinical trial data, adequate supportive care supplemented with different combinations of drugs remains the main treatment for patients. 14, 41 for mers, despite many studies in humans, there is no consensus on the best treatment; thus, randomized clinical trials are needed to assess potential treatment options. 134 several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn and novel therapies, including polyclonal antibodies and broad-spectrum antivirals. 108 antimicrobial peptides (amps) may be used as alternative therapeutic agents, 135 and many have been effective even against bacterial proteases agents. 136 antiviral drugs with effective activity in vitro include neutralizing monoclonal antibodies, antiviral peptides, mycophenolic acid, lopinavir, ifn, ribavirin, nitazoxamide, mycophenolate mofetil (mmf), alisporivir, silvestro, and corticosteroids. 19, 21, 59 although there is no agreement on the most ideal treatment option to cure covid-19, much research activities and pursued routes are underway. potential host-targeted agents the host-directed strategy is another approach for limiting viral replication. host proteases such as cathepsin b and cathepsin l can cleave the spike protein of sars-cov. drugs such as camostat inhibit host serine proteases and then interfere with the entry of sars-cov, but they may cause many significant side effects. 14, 135 pegylated ifn α-2a and -2b may be used to stimulate innate antiviral responses in patients, and chloroquine, an approved immune modulator, was reported to have inhibitory effects against sars-cov-2 under certain conditions. 34 researchers have also proposed that some commercially available drugs with suitable safety profiles, such as metformin, glitazones, fibrates, sartans, and atorvastin, as well as nutrient supplements and biologics, might reduce immunopathology, boost immune responses, and prevent or curb ards. in addition, ongoing cellular therapies using mesenchymal stromal cells from allogeneic donors have been shown to reduce nonproductive inflammation and affect tissue regeneration and are being evaluated in phase 1/2 trials in patients with ards; these therapies may be assayed in sars-cov-2-infected patients. 40, 137, 138 expansion of antiviral t cells as cellular drugs could aid in preparing t cell products for adjunct treatment of patients with severe infection. 40 overall, there are similarities and differences among the treatments for these three coronaviruses. notably, we summarize the potential drugs and vaccines in table 2 . vaccination could be used to prevent infection or to reduce disease severity. different kinds of vaccines, such as dna vaccines, recombinant proteins, subunit vaccines, and inactivated viruses, were described. 14 however, the highly sophisticated immune evasion mechanisms of viral pathogens and their high mutation rates make human vaccine development a major challenge. 136 the four nsps (3clpro, plpro, helicase, and rdrp), which are key enzymes in the viral life cycle, and the s protein, which is responsible for receptor binding during cell entry, are attractive targets for developing vaccines against coronaviruses. 115 among them, the s protein is most commonly targeted. 61 based on previous studies, it is believed that the s protein receptor-binding domain (s-rbd) located in the s1 subunit is an important target for the development of a sars vaccine, especially the key neutralizing region, cnd. indeed, cnd can induce a strong neutralizing antibody response and crossprotection against sars-cov mutants. one study showed that a recombinant fusion protein (designated rbd-fc) containing the 193-amino acid rbd and a human igg1 fc fragment can induce highly potent antibody responses in immunized rabbits. the antibodies inhibited sars-cov infection (serum dilution of 1:10,240), and they are believed to be safer than other types of vaccines. 139 additionally, with chloroplast transgenic technology, it is possible to combine the fusion gene s-rbd and the carrier molecule cholera toxin b subunit into the tobacco chloroplast genome to obtain a chloroplast transgenic tobacco plant that stably expresses the oral sars-cov subunit vaccine. 140 for mers, vaccines based on the viral s protein include full-length s or strimer protein-based vaccines such as a full-length s-based simian adenovirus vector vaccine (chadox1) and dna vaccine (gls-5300), 61 s1-based vaccines such as an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1) and an rabv vector encoding an s1-elicited antibody, 141, 142 rbd-based vaccines, and vaccines based on other regions. it has been confirmed that the s proteins of sars-cov and sars-cov-2 are quite similar, but researchers recently found that the three monoclonal antibodies developed to bind to the s protein of sars-cov, s230, m396, and 80r do not cross-react with the s protein rbd of sars-cov-2, simian adenovirus vector vaccine (chadox1);an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1); an rabv vector encoding s1 elicit antibody; rbd-based vaccines 61, 138, 139, 140 overview of lethal human coronaviruses chen et al. suggesting that tailored vaccines and antibodies against sars-cov-2 must be designed. 143 dna vaccines are also quite promising. specific igg antibodies against sars-cov can be promoted by the s gene dna vaccine. as mentioned above, the s protein of sars-cov plays an important role during the pathogenic process, and synthetic peptides induced by dna vaccine in escherichia coli elicit specific antibodies against the sars-cov s protein which might provide another approach for further developing sars-cov vaccines. 42, 144 to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls-5300) that expresses the s protein of mers-cov, a phase i clinical trial on healthy volunteers was conducted in 2016, but the results were not reported. another phase i trial utilizing the viral vector, chimpanzee adenovirus, oxford university #1 (chadox1), containing the mers-cov s protein expression gene was started by oxford university in january 2018. 145 in addition, camel vaccines against mers-cov are a consideration. at present, at least two promising candidate camel vaccines are undergoing development, and field trial evaluation is in progress. 108, 146 one study found that the rbd fragment covering spike residues 377-588 is a key neutralizing receptor-binding fragment and an ideal candidate for mers vaccines. 147 another potential neutralizing epitope is a peptide fragment covering 736-761 residues of the s protein which blocks the membrane fusion and cellular entry of mers-cov 114 (fig. 5) . other approaches recombinant viruses may be employed to generate an immune response against the viruses. there are two kinds of recombinant viruses which are promising for designing a protective vaccine. the first is a defective or nonpathogenic vector capable of expressing viral proteins, and the second is a vector that can be assembled in a test tube to stimulate the assembly of virus-like particles. adenosine deaminase (ada), a kind of human enzyme, could interact with dpp4, therefore, ada could compete with mers-cov as a natural antagonist when the virus attempts to bind to cells. 148 in addition, anti-dpp4 can play a similar role, 149 however, it is not practical to use this method in vivo because dpp4 has important functions in the regulation of several different signaling pathways. 150 questions concerning the coronavirus-host interactions this topic has intrigued the community. understanding certain mechanisms about virus interactions will support drug research activities. for instance, it was found that sars-cov-2 has a stronger, more rapid spreading ability than many known viruses. is it possible that it invades host cells via additional novel routes, not limited to ace2? for example, cd147 is probably involved in virus invasion. 82 is there an s-like protein involved in invasion? the affinity of the human ace2 protein for the rbd of the new coronavirus is 10-20 times higher than it is for that of the sars virus, which likely explains why sars-cov-2 spreads more swiftly. there is a distinct insert present in the peptide fragment spanning of s1/s2 loop of the sars-cov-2 s protein, which is not shared the similarity with sars-cov or any sars-related viruses. does this peptide loop impact on the entry pathway type of sars-cov-2, compared to other known betacoronavirus lineage b? furthermore, the s protein is likely cleaved during virus assembly and delivery to the cell surface by golgi-resident proprotein convertases such as furin, 151 which is different from the behavior of its close relatives. furin is found in a variety of human tissues, including the lungs, liver, and small intestine. therefore, which are exactly the cell types that sars-cov-2 may attack? yet, in relation to the last two questions, it should be noted that studies are also necessary to investigate any possibility of receptor-independent virus entry. the sars-cov-2 genome encodes nonstructural proteins, structural proteins, and accessory proteins. 3clpro, plpro, helicase, and rdrp, which belong to the first type, and the s protein, which belongs to the second, have been recognized as promising targets for developing antiviral agents against sars-cov and mers-cov, which therefore may be applied to sars-cov-2. nonetheless, the rapid identification of effective interventions against sars-cov-2 is a major challenge. however, the subject of using currently known antiviral drugs has been frequently brought up by the community as a potential short-term strategy to combat sars-cov-2. drugs affecting such targets and their status for sars-cov-2 are listed below. the main candidates as inhibitors of 3clpro include other agents for sars-cov-2 fig. 5 the targets of the different drug candidates against the three coronaviruses. common targets against the three coronaviruses are mainly the s protein and the s1/s2 subunits, pl protein, rdrp, 3cl protein, and helicase. the figure shows drug candidates (in black) and vaccines (in red). among them, remdesivir has been trending in the news recently. it inhibits the rdrp, is in phase iii for sars-cov-2, and may have an effect on the three viruses. ribavirin in combination with a pegylated interferon may also have an effect against the three viruses. ritonavir and lopinavir, which inhibit the 3clpro and are in phase iii for sars-cov-2, have an effect on both sars-cov-2 and mers-cov. dna vaccines and vaccines based on the s protein or subunits of the s protein are in development lopinavir, ritonavir, darunavir, cobicistat, and asc09f (phase iii, in combination with oseltamivir, for sars-cov-2). 152 candidates as inhibitors of plpro include thiopurine analogs, compound 6 (preclinical), and remdesivir (phase iii for mars-cov). 121, 153 favipiravir, ribavirin (randomized trial for sars-cov-2), and remdesivir are among the candidate inhibitors of rdrp. 118 previously, compounds that may interfere with atpase and helicase activities were reported (before covid-19), such as bananins, 5-hydroxychromone derivatives, and triazole derivatives (preclinical). 34, 78 candidates that may suppress viral entry by targeting the s protein include peptide p9 and α-helical lipopeptides (preclinical), 154 and those targeting the s2 subunit of the s protein mainly include hr1p, hr1m, hr1l, hr2l, hr2p, and hr2l (preclinical). 155 due to the strong affinity between ace2 receptor and the rbd of sars-cov-2, another opportunity might be through the development of antibodies to block ace2. the second method is to use a large amount of soluble ace2 to directly block the spike protein. the third method is to find a drug that directly inhibits the spike membrane fusion process, such as the aids drug enfuvirtide. the fourth approach is to identify an agent that inhibits the activity of disintegrin and metalloproteinase 17 (adam17), which may also prevent viral release and proliferation in cells and protect ace2 and the lungs. 87 mrna vaccine technology sars-cov-2 vaccines are already under development. the mrna and dna vaccines are promising approaches to prevent cellular invasion by similar coronaviruses in the future. in january 2020, the coalition for epidemic preparedness innovations (cepi) announced three partnerships to develop a new coronavirus vaccine. among them, moderna and inovio presented mrna and dna vaccine technologies, respectively. the goal of the three teams is to have at least one candidate vaccine capable of preventing coronavirus infection in 16 weeks, to be tested in a phase i clinical trial. moderna's vaccine model is designed to use mrnas to safely pre-expose the immune system to a small amount of encoded proteins usually generated by the pathogen, so that the immune system becomes prepared to fight future pathogens and prevent infection. the candidate genes developed by moderna contain mrnas, and combining multiple mrnas into a single vaccine might be useful to rapidly induce responses, which is an approach that may be applied for future, emerging pandemic threats too. at the same time, because the mrna in the cell will be degraded over time, the mrna vaccine will not continue to produce antigen components for a long time, which can avoid the risk of continued stimulation of the immune system. generally, mrna vaccines are also described as simple to prepare and to yield remarkable results. additional advantages, as well as disadvantages, of nucleic acid-based vaccines were described in detail. furthermore, the basis of traditional vaccines, proteins or polysaccharides, cannot be straight forward applied at present against many pathogens. therefore, mrna vaccines would be suitable for achieving rapid, mass production of emergency vaccines in the event of a major epidemic. however, because rna is easily degraded, inducing cells to absorb mrna quickly is a challenge. in recent years, the method for delivery of mrna into cells has been greatly improved, and stemirna and moderna have adopted nanolipid (lpp or lnp (lipid nanoparticle)) drug-loading technology. however, this is still to be developed for mrnas. furthermore, ensuring safety and effectiveness is another challenge for viral mrna vaccines, and clinical validation will have to be carried out carefully. moderna is a worldwide leader in mrna therapy, with currently nine mrna-type vaccine candidates (e.g., mrna-1273 against sars-cov-2). the national institutes of allergy and infectious diseases (niaid) of the national institutes of health (nih) and the mrna vaccine giant moderna have teamed up to bring the new coronavirus vaccine possibly to phase ii within a few months, and phase iii late this year. simultaneously, in january 2020, the translational medicine platform of dongfang hospital affiliated with tongji university cooperated with stemirna (shanghai) biotechnology co., ltd. to rapidly promote the development of a new coronavirus mrna vaccine. in february 2020, the chinese scientific research team announced that animal testing of the newly developed coronavirus vaccine has begun. if animal testing goes well, this new vaccine will enter human clinical trials within a few months. also in february 2020, zhuhai livanda biotechnology co., ltd. announced that the first batch of new coronavirus mrna vaccine standard samples completed during the spring festival had been delivered to relevant national authorities on for animal testing and efficacy verification. the researchers detected the production of target antibodies in mouse serum at day 12 following immunization. the company claimed that this was also the first time worldwide that new coronavirus vaccines developed based on mrna technology have resulted in antibodies in animals. livanda is currently pushing ahead with the project through extensive cooperation with the academy of military medical sciences, the guangdong provincial institute of supervision, and the macau university of science and technology. the antigen used in its development is the same as that used by moderna. dna vaccine technology dna vaccine technology may have many advantages (e.g., safe, fast, less technical barriers), along with potential limitations too. 156 a phase i human clinical trial of the mers-cov vaccine has proven its safety and effectiveness. 157 the dna vaccine based on the sars-cov s protein developed by the team of barney graham and gary nabel of the us-nih vaccine research center (vrc) has achieved positive results in animal experiments and a clinical phase i trial. 158, 159 inovio pharmaceuticals has accumulated extensive safety and immunogenicity data for mers-cov vaccine studies. because of the high degree of similarity between mers-cov and sars-cov-2, lessons from such a vaccine development process may be beneficial for the development of a dna vaccine against sars-cov-2. since january 2020, inovio has been collaborating with several experts and companies (some in china), and has currently already begun phase i clinical trials of the dna vaccine ino-4800. china's cansino biologics might be the leader as it was announced it has moved to phase ii testing of a vaccine called ad5-ncov. the latter is currently often being reported as a dna vaccine, but to be precise, it uses viral vectors (adenovirus) to deliver dna related to sars-cov-2. part of the project is carried out currently with the chinese military medical research institute. the vaccine candidate is constructed using genetic engineering methods and defective human type 5 adenovirus as a vector that can express the sars-cov-2 s antigen, to stimulate the body to produce strong humoral or cellular immunity. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, and sars-cov-2 is a new member of betacoronaviruses, distinct from sars-cov and mers-cov. sars-cov and sars-cov-2 are similar in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4 (cd26). all of them evolved a mechanism to escape the host cell immune system. sars-cov and sars-cov-2 affect the ras system by suppressing ace2, leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. current treatments for sars mainly include ifn-α, antiviral treatments (e.g., ribavirin), plasma therapy, host-directed therapies, and systemic corticosteroids. for mers, several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn, and novel therapies, including polyclonal antibodies, broad-spectrum antivirals, and amps. for covid-19, candidates mainly include drugs targeting the s protein, nonstructural proteins (3clpro, plpro, helicase, and rdrp), and viral rna synthesis blockers such as remdesivir and ribavirin. vaccines such as dna vaccine, mrna vaccine, and recombinant vaccines are being rapidly developed. so far this century, human beings have experienced several epidemic outbreaks, and each outbreak had a negative impact at different levels, including health, economy, and even psychology and human behavior. in the future, more precautious measures should be available to guide 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to s1 protein that blocks receptor association potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies exceptionally potent neutralizationof middle east respiratory syndrome coronavirus by human monoclonalantibodies interaction between heptad repeat 1 and 2 regions inspike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors fusion mechanism of 2019-ncov andfusion inhibitors targeting hr1 domain in spike protein the authors gratefully acknowledge the financial support from the national natural science foundation of china (grant no. 31870836), and the 1.3.5 project for disciplines excellence, west china hospital, sichuan university (zyyc20005 to w.c.). competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-312741-0au4nctt authors: lin, panpan; wang, manni; wei, yuquan; kim, taewan; wei, xiawei title: coronavirus in human diseases: mechanisms and advances in clinical treatment date: 2020-10-01 journal: medcomm (beijing) doi: 10.1002/mco2.26 sha: doc_id: 312741 cord_uid: 0au4nctt coronaviruses (covs), a subfamily of coronavirinae, are a panel of single‐stranded rna virus. human coronavirus (hcov) strains (hcov‐229e, hcov‐oc43, hcov‐hku1, hcov‐nl63) usually cause mild upper respiratory diseases and are believed to be harmless. however, other hcovs, associated with severe acute respiratory syndrome, middle east respiratory syndrome, and covid‐19, have been identified as important pathogens due to their potent infectivity and lethality worldwide. moreover, currently, no effective antiviral drugs treatments are available so far. in this review, we summarize the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. f i g u r e 1 genome organization and structure of hcovs human covs generally cause only mild upper respiratory diseases, which is similar to common flu. 11, 12 a novel cov, named sars-cov-2 by the world health organization (who), has emerged again at the end of 2019, causing more infections and deaths worldwide than ever before. 13 the absence of effective antiviral treatments and serious consequences of these three covs have highlighted the urgent need for novel drug development to prevent the spread of covs. herein, this review mainly focuses on the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. covs possess a nonsegmented, positive, single-stranded rna genome of 26-32 kb. 2, 14, 15 all covs have a similar genome arrangement with a 5′-methylated cap structure along with 3′-polyadenylated tail. the replicase gene, occupying about 20 kb, two-thirds of the genome and comprising two open reading frames (orfs), orf1a and orf1b, is located at the 5′ end. 2 it encodes two large polyproteins (pp) 1a and 1ab that can be cleaved by papain-like cysteine protease (plpro) and 3c-like serine protease (3clpro) into nonstructure proteins, involving some proteases, several rna modification enzymes, as well as rna-dependent rna polymerase (rdrp) and helicase (hel) required for virus replication. 16 additionally, an untranslated region (utr) can also be identified at the 5′-end as same as the 3′terminal. structure proteins, encompassing the 3′-terminal one-third of the genome, are arranged in a certain order of hemagglutinin esterase (he) protein that is present in some beta-covs, spike protein (s), small membrane protein (e), membrane protein (m), and nucleocapsid protein (n). in brief, the arrangement of the cov genome can be shown as 5′-utr-replicase gene (orf 1a and orf 1b)-he protein (if have)-s protein-e protein-m protein-n protein-3′ utr-poly (a) 2 ( figure 1 ). cov is named for the club-shaped projections eradiating from the envelope, which forms its corona or crow-like morphology. the shape of the viral particles is roughly spherical with approximately 80-160 nm in diameters. [17] [18] [19] the nucleocapsid protein and the genome rna intertwine to form a helical structure located inside the envelope. for some covs, the spikes on the surface are not only formed by trimers of s protein, but also he proteins. m protein and e protein, two transmembrane proteins, also participate in the composition of the virus (figure 1 ). s protein, a transmembrane protein, mediates the initiation of cov infection by attaching to the specific receptors on the target cells. [20] [21] [22] for a prototypical cov, s protein is usually cleaved into an extracellular receptor binding subunit (s1) and a membrane-anchored subunit (s2), responsible for virus binding and membrane fusion, respectively. 23, 24 two heptad repeats (hr1 and hr2) and enriched alpha-helices are contained in the s2 domain, a feature typical of fusion protein. [25] [26] [27] the receptor-binding domain (rbd) of s protein specifically binds to target receptors, leading to the conformation change of s1/s2 complex that mediates virus entry. 26 furthermore, the rbd also induces potent neutralizing ab response, which turns s protein into an important antigenic determinant capable of protective immunity induction and provides a vital approach for the development of immunotherapies. [28] [29] [30] [31] [32] cov e protein is an integral membrane protein of 76-109 amino acids [33] [34] [35] and is present in small amount in virions. [36] [37] [38] it contains a short hydrophilic n-terminal followed by a single predicted hydrophobic domain and a hydrophilic c-terminal. although its membrane topology remains unclear, cov e protein is commonly referred to as a transmembrane protein with one transmembrane domain. [39] [40] [41] accordingly, the e protein mainly targets er and golgi-complex and participates in part of the life cycle of the virus, including virus assembly, budding, particles release, envelope formation, and viral pathogenesis. 33, 35, [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] in addition, cov e protein may have a crucial role in virus production and maturation, because recombinant covs lacking the e protein exhibit significantly reduced viral titers and toxicity. [51] [52] [53] [54] [55] the m protein is a small transmembrane protein (25) (26) (27) (28) (29) (30) with three transmembrane segments, an n-terminal ectodomain and a c terminal-endodomain, determining the shape of the virion. 56, 57 it is considered as the most abundant structural protein and plays a pivotal role in virion assembly via interacting with other structural proteins. [58] [59] [60] binding of s protein and m protein is essential to the virus assembly and the maintenance of s protein in the er-golgi intermediate compartment (ergic)/golgi complex. [61] [62] [63] virus-like particles (vlps) are assembled when the combination of m and e proteins occurs, which suggests that they are required for the formation of the envelope. 39, [64] [65] [66] additionally, when expressed alone, it can become a homomultimeric complex, the primary driving force of envelope formation. 43, 60, 67 furthermore, as to n protein, a stable nucleocapsid and internal core of virions can be achieved when combined with m protein. 68, 69 the n protein, combined with viral genomic rna to form a helical nucleocapsid inside the viral envelope, is a multifunctional protein. 70 it contains three highly conserved domains: the n-terminal domain (ntd), responsible for rna binding; a c-terminal domain (ctd) that is a hydrophobic and helix-rich terminal, capable of dimerization and oligomerization; and an intrinsically disordered region (rna-binding domain/domain 2) that is a serine/arginine-rich domain (sr-domain) with a significant phosphorylation potential. 15, [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] phosphorylation of the n protein can initiate structural modification leading to an increased rna-binding affinity. 72, 79, 87, 88 the n protein binds to the genomic rna through a beads-on-astring form. likewise, except for the interaction between n protein and nucleic, the ability of complex oligomerization is another pivotal activity required for the formation of the ribonucleoprotein complexes for viral assembly. 89 in addition to the role of the n protein in viral core formation and assembly, 69, [90] [91] [92] it is also involved in other critical processes of viral life cycle such as virus budding and envelope formation, 21,93-95 genomic mrna replication ,and genomic rna synthesis. 96, 97 3 although the pathogenic mechanisms of human covs have not yet been fully understood, the investigation of their unique characteristics of each cov enables to distinguish the various human covs including sars, mers, and sars-cov-2. the crucial early step of the infection of human covs into susceptible host cells is the interaction between viral s protein and cellular target receptors. one of the subunits of s protein, s1, containing rbd, is responsible for specific recognition and binding of the target receptors. the other subunit, s2, is in charge of the membrane fusion. 22, [98] [99] [100] [101] [102] the tissue tropism, as well as the susceptible host species, is mainly determined by the binding of s protein to target receptors. 102 based on lines of known evidence, human covs utilize multiple and different types of cellular receptors rather than use a common receptor. therefore, multiple cellular receptors have been identified as target receptors for the various human covs to date (table 1) . angiotensin-converting enzyme 2 (ace2), the first known human homolog of angiotensin-converting enzyme and the receptor for hcov-nl63, sars-cov, and sars-cov-2, is a vital component of the reninangiotensin system (ras). [103] [104] [105] [106] [107] [108] [109] it is a secreted enzyme with a transmembrane domain, a single metalloprotease active site and a signal peptide, 110 and predominantly expressed in heart, vascular endothelia, epithelia of the small intestine, kidney, and testis; alveolar macrophage and monocytes of the respiratory tract; and epithelial cells of the trachea, bronchi, and alveoli. 103, [110] [111] [112] in contrast to its homolog ace that contributes to the promotion of lung failure pathogenesis, induction of lung edemas, and impairment of lung function, ace2 plays a protective role in severe acute lung injury (ali). imai et al revealed that the deficiency of ace2 in the murine models of acute respiratory distress syndrome (ards) deteriorated the symptom in lung function, which could be recovered by hcov-nl63 hcov-hku1 the recombinant ace2. 113 thus, downregulation of ace2 expression in sars patients could be used as an indicator of severe clinical outcome. 114 besides lung damage caused by sars-cov infection could be attenuated by blocking the renin-angiotensin pathway. 114 overall, ace2 could serve as a novel therapeutic target for severe respiratory diseases. dipeptidyl peptidase iv (dpp4), a type ii transmembrane protein also known as cd26, is identified as a target receptor for mers. 115 it is a prolyl oligopeptidase expressed in various cells including endothelial cells, epithelial cells, and inflammatory cells in the lung and smooth muscle. [116] [117] [118] the multifunctional protein ddp4 is implicated in the activation of t-cell, the activity regulation of chemokines and growth factors, and the regulation of glucose metabolism. [119] [120] [121] [122] not only can it be embedded in the plasma membrane in the form of a homodimer, but it also presents in extracellular fluid like plasma as a soluble form. 118, 123 although ddp4 is expressed in epithelial cells of the upper respiratory tract in camels, it is principally found in alveoli but rarely in the nasal cavity or conducting airway. 115, 124 accordingly, in a murine mers model, though monocyte infiltration, alveolar edema and microvascular thrombosis were observed in the mers-cov-infected lungs, any symptoms were seldom found in the airways. 125 aminopeptidase n (apn), a type of ii metalloprotease also called cd13, is a ubiquitous enzyme expressed in various organs (lung, intestine, and kidney) and cells (epithelial cells, endothelial cells, leukocyte, and fibroblast). 126, 127 it serves as a target receptor for hcov-229e, 128 but not for hcov-oc43. hcov-oc43 shares the same specific target with hcov-hku1, namely 9-o-acetylated sialic acid. 129,130 virus entry is a finely regulated process requiring a series of interactions between the virion and host cell. [131] [132] [133] following the conjunction with the target receptor, cov fuse its envelope with the membrane of the host cell. these processes are forced by the conformational change of s protein, which is triggered by not only the target receptor binding but also ph acidification and proteolytic cleavage led by cell surface or endosomal proteases such as transmembrane protease serine 2 (tmprss2), furin, cathepsin l, elastase, and trypsin. [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] cleavages of s protein are facilitated at two sites: the boundary between the s1 and s2 subunit (s1/s2) and the conserved site upstream of the fusion peptide (s2'). 138, 144 the former one is aimed at releasing rbd from the membrane fusion subunit, and the latter one is important for the exposure of the fusion peptide, hydrophobic in general, which acts as an anchor to target membrane. 138, 145, 146 then the fusion domain adopts two heptad repeats (hr1/hr2) to form a compact coiled-coil conformation called 6-helix bundle or 6hb. 144, 147 through direct interactions with lipid bilayers, the fusion domain disrupts two apposed membrane layers and fuses the viral envelope to host cell membrane. ultimately, the viral genome is successfully released into the cytosol of the target cell. in addition to the viral infection through the plasma membrane, the entry of covs into cells can be accomplished by the endocytic pathway, depending on the virus strains and host cells. 135,144 sars-cov, whose intermediary host is the palm civet, is a highly pathogenic respiratory virus that emerged in 2002, leading to global pandemic that affected more than 8000 people in 29 countries. 148, 149 patients infected with sars-cov initially present "flu-like" syndrome commonly showing high fever, headache, sore throat, myalgia, and dry cough. [150] [151] [152] during the disease progression, ali or ards is developed in a number of patients. 153 pathological manifestations can be described as three phases. the first step is the disturbance of gas exchange in the first week, owing to the extensive edema, shedding of ciliated epithelial cells, and deposition of hyaline-rich substances on the alveolar membrane. in the next step, pulmonary fibrosis occurs that is characterized as the deposition of fibrin at epithelial cells and the alveolar spaces, as well as the infiltration of inflammatory and fibroblasts. finally, fibrosis of lung tissue, collagen deposition, and proliferation of alveolar and interstitial cells represent the final step of disease, about 6-8 weeks. [154] [155] [156] [157] [158] diffuse alveolar damage (dad) accompanied by hyaline membrane formation as well as interstitial thickening is common characteristics of sars-cov inducing pulmonary damage. 159 although many investigators have devoted to inquiring into the pathogenesis of such virus, it has not yet been fully understood until now. the immune response is the earliest alert system of the host cells warning virus attacks. ironically, it also aids viral pathogenesis. pattern recognition receptors (prrs) that are retinoic acid-inducible gene i protein (rig-i, member of rlrs family) and melanoma differentiation-associated protein 5 (mda5) recognize viral pathogen-associated molecular patterns (pamps), such as viral components and replication intermediates, to initiate signaling cascades against virus infection. 160, 161 once the pamps from invaded viruses are detected, rig-i and mda5 interact with the mitochondrial antiviral signaling protein (mavs) that is a mitochondrial membrane-bound f i g u r e 2 escape mechanisms of innate immune response of sars-cov and mers-cov adaptor molecule, followed by the activation of several kinase complexes and multiple subsequent transcription factors (irf3, irf7, and nf-κb). activation of nf-κb induces the production of proinflammatory cytokines and chemokines in the nucleus that is a substantial cause of the ards. 162, 163 phosphorylation of irf3 and irf7 by the kinase complexes results in homo-or heterodimerization of irf3 and irf7. the dimerization initiates the transcription of type i interferons (ifns, ifn-α and ifn-β), activating the signal transducer and activator of transcription proteins (stats) to mediate antiviral response ( figure 2 ). [164] [165] [166] several defensive approaches are used by sars-cov to avoid the prrs defense system and, ultimately, host innate immune response. one approach is to replicate themselves within the double membrane vesicles (dmvs) that are protected from the prrs. 167, 168 the eukaryotic mrnas contain a 5′ cap that usually lacks in the viral mrnas. however, some viruses such as sars-cov are capable of building the rna cap through nsp14 and nsp16/nap10 complex, helping them bypass the recognition by prrs. [169] [170] [171] [172] in addition, a couple of more proteins encoded by the viruses participate in the suppression of the innate immune response by disrupting the ifn response. among the nonstructural proteins, nsp 1 mainly involves in the degradation of host mrnas, inactivation of host translational machinery as well as the inhibition of stat1 phosphorylating. [173] [174] [175] sars-cov plpro interferes phosphorylation of irf3 and disrupts nf-κb signaling probably via interacting with sting. [176] [177] [178] the nsp7 and nsp15 are also potential ifn antagonists though the mechanism is not clear. 177 structure proteins such as the n protein and m protein are likely to suppress the type i ifn path-ways. because it was known that n protein inhibits the ifn transcription, the n protein could have a strong potential influence on the viral rna. 179 m protein blocks the formation of signaling kinase complexes, and suppresses the irf3 and irf7 activities, suggesting the potential role of the n protein in the viral infection as well. 180 such accessory proteins as orf 3b protein are able to inhibit the rig-i activity and irf3 phosphorylation in addition to the transcription of ifn-stimulated genes (isgs) via the isre promoter, while orf6 blocks the nuclear translocation of stat1. 181, 182 in spite of the number of research findings in vitro, they have not been validated in vivo. therefore, it is urgent to examine the findings in vivo for a clear and solid understanding of the infectious process. besides the immune response of the host, ace2 also plays an essential role in the pathogenesis of sars-cov. as a negative regulator on the ras, ace2 has been closely linked to the pathogenesis of pulmonary diseases and considered as a protective factor for ali. 113 consequently, the downregulation of ace2 mediated by sars-cov binding might give an explanation for the progression of severe lung damage occurred on some sars patients. 114 a decade after sars, another novel cov was identified as the pathogen of mers that caused a higher mortality rate (30%-40%) compared with sars (around 10%). 183, 184 sars-cov and mers-cov are both emerged from bats, and are disseminated to human through palm civets and dromedary camels, respectively. [185] [186] [187] [188] mers-cov and sars-cov share some common clinical manifestations ranging from asymptomatic to severe pneumonia in multiple organs, 150, 184, 189 and pathological features including inflammatory cells infiltration and dad. 183 similar to sars-cov, mers-cov is also capable of causing immune dysregulation by attenuating the innate immune response ( figure 2 ). 190, 191 type i interferon is important for the inhibition of mers-cov replication in host cells probably via the suppression of the dmvs formation. 192, 193 capping viral mrna by nsp14 and nsp16/nap10 complex protects mers-cov, as well as sars-cov, from the prrs recognition since the structure of nsp16/nap10 complex in both viruses are analogous. 194 besides, several proteins of mers-cov are involved in immune escape mechanism by involving in the signaling cascades. it was reported that irf3 nuclear translocation and ifn promoter activation are inhibited by m protein, orf4a, orf4b, and orf5, former three of which also restrained the expression from an the isre promoter. 195 inhibition of the phosphorylation of irf3 might be the mechanism for ifr3 translocation inhibiting. 194 moreover, mers-cov orf4a can interact with ifn-inducible double-stranded dna (dsdna)dependent protein kinase activator a (prkra) and subsequently control the function of rig-i and mda5, resulting in the disruption of the ifn response. 196, 197 orf4a and orf4b are thought to participate in the nf-κb signaling by downregulating the gene stimulated by nf-κb and affecting the kinase complexes, respectively. 198 functions of plpro and nsp1 of mers-cov are analogous to the functions of those in sars-cov. 199, 200 like ace2, the entry receptor dpp4 for mers-cov has also a pivotal role in the disease pathogenesis and is considered as a key factor for the intraspecies variation shown in mers infection. 201, 202 it is usually expressed in type ii pneumocytes that cover 2% of the alveolar surface. 115, 124 approximately 95% of the surface area is occupied by the type i pneumocytes that are responsible for gas exchange. 203, 204 but the autopsy reports indicated that both type i and ii pneumocytes in patients died from mers-harbored dpp4 expression and these pneumocytes were infected by the virus. 205, 206 mers-cov infection of type i pneumocytes might lead to the damage of the cells in the alveoli subsequently causing the dad. 207 it suggests that type i pneumocytes expressing dpp4 might be included in the pathogenesis of the disease. this might explain why chronic obstructive pulmonary disease (copd) patients attacked by mers-cov had poor outcomes, since the expression of dpp4 was predominantly upregulated on type i pneumocytes in such patients. 202, 208 besides, owing to high expression of dpp4 shown in the kidney, the renal dysfunction might be caused by either the direct infection or the hypoxic damage. 116 evidence of tubular injury, such as cell debris and tubular dilation, could be observed in the late stage of the infection in mers-cov-infected mice. however, no virus could be detected in such animals in the early stage after infection, meaning such pathologic changes might be related to the hypoxia. 125 the outbreak of covid-19, whose pathogen is sars-cov-2, now poses a serious threat to the global public health. 209, 210 since the emergence of the virus, sars-cov-2 has affected more than 14 million people with more than 597 thousand deaths worldwide as of july 2020. next-generation sequencing of the novel virus has been developed soon after the outbreak, indicating that sars-cov-2 is closely related to the bat-derived sarslike covs. 107, 211 it is now believed that bats are likely to be the natural reservoir, 212, 213 and pangolins are regarded as intermediary host according to the later studies. typical clinical presentations of sars-cov-2-infected patients include fever, dyspnea, dry cough fatigue, myalgia, pneumonia, and ards symptoms, similar to those of sars and mers patients. [214] [215] [216] however, intestinal disorders such as diarrhea are less frequent in covid-19 patients than sars. 214, 215, 217 furthermore, in spite of the variation of amino acid at some residues, homology modeling informed that sars-cov-2 and sars-cov have an analogous rbd structure, and share the same target cell receptor, ace2, to mediate the viral entry. 107, 108, 216, [218] [219] [220] it is speculated that ace2 is involved in the pathogenesis of sars-cov-2. owing to the current sparsity of data on the pathological characters of sars-cov-2, it is poorly understood. a case report from an infected patient died of this disease showed that dad with hyaline membrane formation, infiltration of inflammatory cells, and pulmonary edema could be found in the samples taken from their lungs, which is notably corresponding with the symptoms of sars and mers patients. 221 additionally, lymphopenia is a common manifestation in covid-19 patients and might be an effective indicator to estimate the severity of hospitalized covid-19 patients. 222 lymphopenia is also supposed to be a vital factor related to the pathogenesis that has not been elucidated so far. 213 moreover, the concentration of some cytokines and chemokines detected in the plasma was higher in covid-19 patients compared with healthy individuals. moreover, higher plasma levels of gscf, il-2, il-7, il-10, mcp1, mip1a, ip10, and tnf-α were linked to the more severe disease. 215 all of the data reveal that immunopathology may occupy a crucial place in the development of the disease, and further researches about the pathogenesis of sars-cov-2 are urgently needed in the future. owing to the fact that no effective specific antiviral therapies are currently available for sars, mers, and covid-19, isolation and symptomatic support cares are the major management strategies for suspected and confirmed cases requiring hospital treatment including oxygen inhalation, fluid management, and rational use of antibiotics, to prevent organ failure and secondary bacterial infection and alleviate the symptoms. 189, [223] [224] [225] thus, the identification of effective agents against human covs is urgently needed in the response to the current covid-19 outbreak. all of sars-cov, mers-cov, and sars-cov-2 encode structure proteins (like s protein), nonstructure proteins (eg, plpro, 3clpro, rdrp, and helicase), and accessory proteins that are essential for the viral life cycle and that are considered as important targets for the development of antiviral agents ( figure 3 ). 226, 227 analyses of genomic sequences and protein structure indicated that the catalytic sites of four crucial enzymes and the key drug-binding pockets in viral enzymes were conserved across sars-cov, mers-cov, and sars-cov-2. 227 therefore, the therapeutic experience based on sars and mers is capable to guide the treatment of covid-19. the idea to disturb the normal life cycle of the virus provides significant insights into the clinical treatment strategy. the s protein is important for the discovery of antiviral agents due to its multifunction in virus infection. rbd located on the s1 subunit can bind to the host cell receptors (ace2 for sars-cov and sars-cov-2, dpp4 for mers-cov) initiating the conformational changes in s2 subunit to get viral and cell membranes closer and trigger membrane fusion. 228 consequently, the interaction between rbd and the host cell receptors serves as a key target for the production of neutralizing antibody followed by the vaccine development. 31, 229 monoclonal antibodies (mabs) and fusion inhibitors against s1 and s2 subunit, respectively, are potential antivirus drugs to conquer the viral infection, and the agents targeting the host receptors also play a similar role. [230] [231] [232] [233] [234] likewise, cleavage at the protease site of the s1/s2 complex by host proteases such as tmprss2 and furin is necessary for the membrane fusion and syncytium formation. 143, 235 the endosomal cysteine protease cathepsins promote the entry of covs into the host cell via the endosomal pathway. 236 inhibitors of these host proteases can potently block the cell entry, which has been proved in vitro and require further validation on animals studies. 237 once entering the host cells, covs release the nucleocapsid and genomic rna into the cytoplasm and start the translation of the replicase gene. the large replicase pp1a and pp1ab are cleaved by plpro and 3clpro to produce nonstructural proteins like rdrp and helicase, forming the replication-transcription complex. 238 numerous agents inhibiting these proteins have shown anti-cov effects in vitro. combination of the hydrophobic domains of the replication-transcription complex to the endoplasmic reticulum membrane can form the typical cov replication structures such as dmvs and convoluted membranes, protecting covs from the detection of prrs. 168, 239 viral rna synthesis produces genomic and subgenomic rnas. then the subgenomic rnas are translated to generate the structural and accessory proteins, participating in the assembly of the virion that is released into the extracellular compartment via exocytosis. 8 small interfering rnas (sirnas) disturbing these processes could be used in the treatment of covs infections. although covs are capable of disturbing the ifn response, they are still sensitive to the ifn treatment in vitro, indicating that augmented host innate ifn response can be an effective strategy to control the viral infection. 207, [240] [241] [242] in addition to the enhancement of inf response, several other cell signaling pathways are also regarded as potential anti-cov targets. these include calcineurin-nuclear factor of activated t cells (nfat) pathway and kinase signaling pathways such as erk/mapk and pi3k/akt/mtor pathways, the inhibitors of which have exhibited anti-cov activities as well. [243] [244] [245] since the discovery of new interventions may take months or even years, a more efficient approach is to repurpose existing antiviral agents approved for treating related viral infections. the followings are approved drugs or preclinical compounds that have potential antiviral abilities against sars, mers, and covid-19. virus-targeted therapeutic strategies all of the major proteases of the virus are attractive druggable targets since they are essential for viral transcription and replication ( table 2) . as a key part of replication-transcription complex, rdrp participates in the production of genomic rna and subgenomic rna. nucleoside analogues targeting rdrp is capable of inhibiting viral rna synthesis in a great variety of rna viruses including covs. [246] [247] [248] [249] [250] favipiravir (t-705), a guanine analogue approved in japan for influenza treatment, has been proven to effectively interfere the rna synthesis of rna viruses such as influenza virus, ebola virus, and other hemorrhagic fever viruses at rdrp level. [251] [252] [253] [254] [255] [256] several studies concluded that favipiravir could inhibit avian influenza a (h5n1) virus and ebola virus infection in mice. 256, 257 also, favipiravir has been proved with a notable effect increasing the survival rate of ebolainfected patients from 35.3% to 56.4% in sierra leone. 258 a recent study ended with the statement that favipiravir owns the ability against sars-cov-2 (ec50 = 61.88um, cc50 > 400um, si > 6.46). 259 covid-19 patients were enrolled in randomized trials for the evaluation of the efficacy of favipiravir plus inf-α or baloxavir marboxil (chictr2000029544 and chictr2000029600). another guanosine derivative with broad-spectrum antiviral activity, ribavirin, has been authorized for hcv and respiratory syncytial virus (rsv) treatment. 260 accurate mechanism of ribavirin against virus infection is not clear, but inhibition of mrna capping and viral rna synthesis could be pivotal to its antiviral activity. 261 although high dose of ribavirin has been applied to sars treatment, the anti-mers-cov effects were moderate at such dose in rhesus macaques infected by mers-cov and no obvious survival benefit has been observed in mers patients. 225, [262] [263] [264] [265] [266] [267] recently, an open-label, randomized phase ii clinical trial (nct04276688) has revealed that triple combination of ribavirin, interferon, and lopinavirritonavir in covid-19 treatment was safe and superior to lopinavir-ritonavir therapy alone in remission of symptoms, shortening virus shedding and promoting discharge of mild to moderate covid-19 patients. 268 remdesivir (gs-5734) is a small-molecule monophosphoramidate prodrug of an adenosine analog with the ability to interfere with the rna polymerase and the proofreading exoribonuclease and terminate the nonobligate chain. 269 on day 1 and 100 mg once-daily maintenance for 9 days. however, the first clinical trial (nct04252664) has been suspended so far and the second trial (nct04257656) with 237 covid-19 patients enrolled finally indicated that remdesivir hardly shown any statistically significant clinical benefits. 274 conversely, a research found that 36 of 53 (68%) hospitalized patients suffered from severe covid-19 and treated with compassionate-use remdesivir could achieve clinical improvement. 275 in addition, a phase iii, randomized, double-blind, placebo-controlled trial (nct04280705) conducted by beigel et al uncovered the fact that remdesivir prevailed over placebo in shortening the time to recovery in adults patients. 276 though remdesivir has been approved by the food and drug administration (fda) to treat severe covid-19 patients, further researches are urgently required to determine the efficacy and the indication of remdesivir therapy. a novel synthesized nucleoside analogue, bcx4430 (galidesivir), is designed to inhibit viral rna polymerase activity via terminating nonobligate rna chain. 277 bcx4430 exhibits a promising antiviral capability against a wide array of . 277 it is currently tested in phase i clinical trial (nct03800173) for marburg virus and can be a potential countermeasure against viral diseases that threaten the public health in the world. a recent study also concluded that penciclovir, another rdrp inhibitor that is approved for hsv, showed effects on reducing sars-cov-2 infection by high-dose administration (ec50 = 95.96 µm, cc50 > 400 µm, si > 4.17). 259 although resistance to nucleoside analogs has rarely been reported, it is worth noting that mutation in rdrp is probably responsible for the acquired resistance and should be monitored for the possible resistance. 278 covs plpro enzymes display proteolytic, deubiquitylating, and deisgylating activities. [279] [280] [281] plpro was first regarded as a druggable target for sars-cov, and then several compounds targeting sars-cov plpro were also found to be effective against mers-cov plpro, recently. 282, 283 though numerous plpro inhibitors have been identified, many of them only inhibit enzymatic activities and do not affect on the viral replication. 284, 285 a study from lin et al suggested that an fda-approved alcoholaversive drug, disulfiram could inhibit sars-cov and mers-cov plpro via different mechanisms. and the synergistic inhibition between disulfiram and known plpro inhibitors, like 6-thioguanine and mycophenolic acid, to mers-cov might offer the potential combination treatments against covs in clinical. 286 another essential protease that cleaves the viral polyproteins during viral replication is 3clpro. similar to plpro, a great many of inhibitors have been identified with the ability to target covs 3clpro. among the 3clpro inhibitors, the human immunodeficiency virus (hiv) protease inhibitors are the most comprehensively studied such as lopinavir, ritonavir, asc09f, as well as darunavir and cobicistat. lopinavir and ritonavir, applied in combination to treat hiv infection, have shown improvement in the outcome of sars patients in nonrandomized trials. 287, 288 though lopinavir alone hardly displayed antiviral activity against mers-cov in vitro, the combination of lopinavir and ritonavir ameliorated the outcome in mers-cov-infected nonhuman primates. 289, 290 therefore, the efficacy of the lopinavir-ritonavir combination in mers patients should be reappraised (nct02845843). however, no benefit was observed in lopinavir-ritonavir treatment compared to standard care in a randomized, controlled, open-label clinical trial (chictr2000029308) involving severe covid-19 patients. 291 further trials are still needed to confirm the therapeutic efficacy. in addition, several other clinical trials have been developed to confirm the efficacy of 3clpro inhibitors on covid-19 (nct04252274, nct04251871, nct04255017, chictr2000029539, nct04251871, nct04255017, and nct04261270), as well as darunavir and cobicistat (nct04252274), asc09f combined with oseltamivir (nct04261270). helicase acts on the duplex oligonucleotides and turns them into single strands in an atp-dependent manner in the cov replication cycle. that helicases in different covs are highly homologous making them potentially strong targets for the covs therapeutic options. based on the mechanism actions, the helicase inhibitors can be approximately classified into two groups. one is able to inhibit both the atpase and helicase activities represented by bananins derivatives, 5-hydroxychromone derivatives, and triazole derivatives (compound 16). 292, 293 the other group including ssya 10-001 and adks has the ability to inhibit the helicase activity with no or little effects on the atpase activity. 294 however, the toxicity of helicase inhibitors needs to be examined before being applied to human patients. the transmembrane glycoprotein, s protein, is also a promising target for antiviral agents' development ( table 2 ). one class of antiviral drugs targeting s protein mostly blocks the spike-mediated membrane fusion. a potent mers-cov inhibitor, nafamostat, has demonstrated to be inhibitive against the sars-cov-2 infection (ec50 = 22.50 µm, cc50 > 100 µm, si > 4.44). 259 griffithsin is a red-alga-derived lectin, which specially binds to oligosaccharides located on the surface of viral glycoproteins, for example, s glycoprotein of sars-cov and hiv glycoprotein 120. 295 a wide range of human covs infection could be inhibited by griffithsin, comprising hcov-229e, hcov-nl63, hcov-oc43, and sars-cov. 295, 296 but the value of griffithsin in the treatment or prevention of covid-19 is needed to be evaluated urgently. s2 subunit of s protein contains two heptad repeat (hr1 and hr2). antiviral peptides analogous derived from these regions exhibited inhibition to the spike protein-mediated cell-cell fusion and viral entry in viruses such as sars-cov, mers-cov, as well as hcov-229e. 231, 297, 298 hr2p, a peptide spanning hr2 sequences of mers-cov s protein, was capable of interacting with hr1 region to form a 6-hb complex, resulting in potent inhibition of viral fusion and replication. its analog hr2p-m2 exhibited an obvious improvement in stability, solubility, and antiviral activity after being modified with hydrophilic residues. 228 additionally, hr2p-m2 intranasal administration effectively prevented experimental mice transduced by adenoviral vectors conveying human dpp4 from mers-cov infection with a >1000-fold decrease in viral titers in the lung, and this protection was intensified via the combination of hr2p-m2 and ifn-β. 299 another newly designed fusion inhibitor from mers-cov called mers-five-helix bundle (mers-5hb), which is derived from the 6-hb, also displayed a potent suppression on s protein-mediated syncytial formation. compared with mers-6hb, mers-5hb lacks one hr2, which endows its capability to interact with viral hr2 to interrupt the membrane fusion. 300 besides, mers-5hb could effectively inhibit the entry of pseudotyped mers-cov with 50% inhibitory concentration (ic50) about 1 µm. 300 altogether, the resistance of these drugs can be overcome by combining antiviral peptides aiming at various domains of s2 subunit, which may also attain synergistic effects in vitro. as for sirnas, which displayed antiviral activities in vitro as well as in sars-cov-infected rhesus macaques, are still under preclinical development and demand further studies to seek out the reliable drug delivery methods in a human before the clinical application. [301] [302] [303] m, n, and e proteins and some accessory proteins are not only vital to the virion assembly but also involved in viral pathogenesis in which they function in the interruption of host innate immune response to assist viral infection. for instance, m protein as well as accessory proteins 4a, 4b, and 5 of mers-cov act as ifn antagonist, and n protein of sars-cov serves as an inhibitor of viral rna. researches carried out by he et al and akerstrom et al emphasized that sirnas silencing m, n, e, orf3a, and orf7a/7b play an important role in the inhibition of viral replication of the sars-cov. 304, 305 but the delivery methods still limit their application in human being. nevertheless, various agents related to these proteins are discovered via functional analysis. one example is resveratrol, a natural stilbene derivative demonstrated to reduce inflammation and exert antiviral activity against multiple viruses. [306] [307] [308] [309] [310] [311] in addition, it exhibited significant inhibition of mers-cov infection and prolonged cellular survival after virus challenge in vitro via deregulating the expression of n protein and the apoptosis induced by mers-cov. 312 alternatively, hexamethylene amiloride, a viroporin inhibitor, is capable to suppress the ion channel activity of e protein of covs such as hcov-229e and sars-cov. 313 identified as dna metabolism inhibitor, gemcitabine hydrochloride is a deoxycytidine analog inhibiting both sars-cov and mers-cov with micromolar ec50 and low toxicity, which suggests its potential antiviral capacity for other human covs. 314 lj001 and jl103, two novel lipophilic thiazolidine derivatives, could induce several changes in membrane properties including the decrease in membrane fluidity, contributing to inhibition of membrane fusion, which made them become broad-spectrum enveloped virus entry inhibitors and potential anti-cov agents. 315 host-cell-targeted therapies the host innate immune response is vital to the interruption of viral replication. recombinant interferons have been applicated in treating various viruses as well as many covs (table 3) . though the host interferon response can be inhibited by the covs, they are still proved effective against covs infection such as sars-cov and mers-cov in vitro and several animal models. 242, 264, 289, 299, 316 recombinant interferons are usually combined with other antiviral agents including ribavirin or lopinavir/ritonavir to treat sars-cov and mers-cov infections, 290, 317 and the anti-covs efficacy of interferons is enhanced when added with ribavirin. 318 a combination of interferon-α2b with ribavirin reduced virus replication and improves clinical outcomes in a rhesus macaque model of mers. 264 however, the effectiveness of combination treatments comprising interferons and ribavirin is still controversial in clinical researches. a study of five patients received interferon-α2b and ribavirin showed no survival, but the finding might be not reliable owing to the delayed administration. 266 contrarily, in another study (n = 44), mortality rates of individuals receiving interferon-α2b and ribavirin exhibited a significant reduction in day 14, compared with the individuals received standard supportive care, but no significant difference was observed in day 28. 265 moreover, no significant difference in mortality rates between interferon-α2b and ribavirin treatment group and interferon-β1a and ribavirin ta b l e 3 host-targeted agents for hcovs treating group was observed in a retrospective study. 267 on the other hand, interferon-β1b displayed stronger inhibition to the mers-cov replication in vitro compared with other interferons, and combined use of interferon-β1b with other antiviral compounds should be evaluated in further research. 289, 290 nitazoxanide, originally identified as an antiprotozoal agent, was later considered as a broad-spectrum antiviral agent able to inhibit the replication of numerous dna and rna viruses such as rsv, parainfluenza, rotavirus, hbv, hcv, hiv, yellow fever, as well as covs in vitro. 319 several clinical trials have confirmed its potential value in treating influenza, chronic hbv, and hcv. [319] [320] [321] moreover, recent research indicated that nitazoxanide was capable of inhibiting sars-cov-2 infection at a low micromolar concentration (ec50 = 2.12 µm; cc50 > 35.53 µm; si > 16. 76) , and the in vivo evaluation of this efficacy is demanded. 259 another type i interferon inducer, polyinosinic:polycytidylic acid (poly(i:c)), is an analog of dsdna with a powerful ability to reduce mers-cov load in animal models. 192 and phase ii clinical trials demonstrated that it was well tolerated by malignant gliomas patients when injected intramuscularly. 322, 323 overall, the use of interferons or interferon inducers may be a valuable strategy against covs infection and should be furtherly accessed in animal models. several host factors are considered essential to covs entry, such as the host receptors that bind to covs s protein, and host proteases that facilitated covs entry through the cell surface or endosomal pathway. thus, these factors become attractive targets for anti-cov agents' development (table 3) . antibodies, peptides, and some functional inhibitors targeting the host receptors can effectively interrupt the binding between s protein and the host cells. one example is that the n-(2-aminoethyl)-1-aziridineethanamine (naae), a small molecular inhibitor, is able to target ace2 leading to the block of s protein-mediated membrane fusion, so does the synthetic peptides analogous, p4 and p5. 324, 325 similar agents were also found in mers treatment, one of which, ys110, was confirmed to be well tolerated in patients with advance solid tumors. 326 owing to their specificity to appointed receptors, they were regarded as narrow-spectrum drugs. however, the efficacy of these receptor-targeted agents have never been evaluated in any covs-infected patients and the safeties of these agents also remain unclear. host protease such as cathepsins and tmprss2 play a key role in the cleavage of s protein and the suppression of these proteases with potent inhibitors can obstruct the virus entry through either endosomal pathway or cell surface pathway. k11777 is a cathepsin inhibitor with broad spectrum against enveloped rna virus including sars-cov and mers-cov. 237, 327, 328 however, the camostat mesylate, applied in chronic pancreatitis treating, is a tmprss2 inhibitor that interrupts the tmprss2mediated cell surface entry. 329, 330 the combination of cathepsin inhibitors and tmprss2 inhibitors is crucial to the complete suppression of mers-cov in vitro. inhibition of another host protease, furin, which is vital to furinmediated s cleavage for covs, also can block membrane fusion and the viral entry like mers-cov. 143 notably, inhibition of host proteases may result in some side effects and need further evaluation. some approved antipsychotic agents (chlorpromazine, triflupromazine, and fluphenazine) and cardiotonic steroids (ouabain and bufalin) can inhibit clathrinmediated endocytosis via affecting the assembly of clathrin-coated pits at the plasma membrane and binding sodium/potassium-transporting atpase subunit α1 (atp1a1), respectively. 314, 331, 332 thus, they are considered as clathrin-mediated endocytosis inhibitors. alternatively, endosomal acidification also has a profound impact on endocytosis. increase of endosomal ph shows an inhibitive effect on virus infection, which has been confirmed by chloroquine, a widely used autoimmune disease and antimalarial agents. 333 chloroquine proves to be active against a wide range of rna viruses including hcov-229e, hcov-oc43, sars-cov, and mers-cov. [334] [335] [336] [337] [338] recently, chloroquine is identified to function at both entry and postentry phase of the sars-cov-2 infection with the ec90 value of 6.90 µm in vero e6 cells. 259 additionally, as an immunoregulator, its antiviral activity may be synergistically intensified in vivo. 259 therefore, chloroquine is suggested as a potential option against the emerging sars-cov-2. significantly, higher dose of chloroquine should not be recommended for severe covid-19 patients owing to the its drug safety, particularly while simultaneously accepting azithromycin and oseltamivir treatment, which was presented by a randomized clinical trial (nct04323527). 339 hydroxychloroquine, a chloroquine analog with lower toxicity, was listed as a potential anti-sars-cov-2 agent and recommended to be applied in covid-19 treatment by chinese national guideline and fda. 340 however, evidence of the benefits and harms of hydroxychloroquine therapy is still insufficient and conflicting. some small studies show that hydroxychloroquine was capable of decreasing sars-cov-2 shedding that could be enhanced by the combination of azithromycin and achieving a shorter time to clinical recovery. 341, 342 but almost no clinical benefit was observed in other studies. 340, 343 therefore, therapeutic efficacy of hydroxychloroquine is still needed to be reconfirmed. moreover, there are several factors required to be reconsidered before efficacy evaluation, such as the severity of illness, definition of the endpoints, and effects of the comorbidities. except for the innate immune response, host receptors, and other factors affecting the endocytosis, some signaling pathways have also been suggested as useful approaches for discovering anti-cov reagents (table 3) . cyclophilins are peptidyl-prolyl isomerases with physiological functions showing as modulating the calcineurin/nfat pathway via reacting with covs nsp1, which is important for the immune cell activation. 244 in addition, they are also required for the replication of numerous viruses including hiv, hcv, as well as covs. 344 consequently, inhibition of cyclophilins by cyclosporines, such as cyclosporin a (csa) and its derivatives, has shown to block the replication of a wide range of covs. 244, 245, 345, 346 however, the obvious immune suppressive properties of csa limit its application in antiviral therapy. but alisporivir, a nonimmunosuppressive cyclosporin a-analog, also displayed the inhibition to the covs replication at a lowmicromolar concentration. 347 additionally, the combined use of cyclosporine and interferon or ribavirin in vitro was beneficial to inhibit sars-cov or mers-cov infection, which needed to be furtherly evaluated in animal models. 347, 348 furthermore, some antiviral agents inhibiting the cellular signaling pathways, in particular, the erk pathway and pi3k/akt pathway, also interrupt the replication of covs. 243, 314, 349 however, the efficacy and safety against covs infection of these agents still need to be reconsidered. corticosteroids, which were used in sars and mers treatment, have been linked to several complications such as psychosis, diabetes, and avascular necrosis. 350, 351 they also were pointed out to be associated with viral replication prolongation in mers patients. 351 however, an update on the efficacy of dexamethasone based on a press release publicized recently indicated that severe covid-19 patients given 6 mg dexamethasone once daily shown a lower mortality (about 8-26%) compared to patients with standard care. 352, 353 besides, another agent, methylprednisolone, also exhibited potential capacity in reducing the mortality rate and achieving better clinical outcomes in severe covid-19 patients. 354, 355 thus, it is wise to apply corticosteroids to the right patients at the right time. but physicians also need to monitor the corticosteroid-related complications. clinical trials of corticosteroid treatments are shown in table 3 . potential immunotherapeutic options s protein of sars-cov proves to be highly immunogenic during infection and responsible for eliciting a humoral immune response in the host. 356 antivirus antibodies could be detected in the plasma of convalescent patients' infected sars-cov and mers-cov. 357, 358 convalescent plasma therapy has been applicated in treating patients infected by numerous viruses involving ebola virus, junin virus, machupo virus, and lassa fever. [359] [360] [361] [362] as for sars-cov, higher day-22 discharge rate and lower mortality rate have been observed among sars patients who received convalescent plasma transfusion before day 14 of the illness. 363, 364 this is consistent with another small cohorts research concluding infected patients with severe conditions who failed to respond to current therapies but finally survived after transfused with convalescent plasma, with no obvious side effects. 358 similar results were found in mers patients. 365 additionally, plasma adoptive therapy with anti-mers-cov antibodies could block the virus adhesion and accelerate the viral elimination from mers-cov-infected animal models. 192 but the efficacy and safety of convalescent plasma therapy in covid-19 patients still need to be reevaluated. although convalescent plasma therapy proves to be a potentially effective therapeutic option for emerging covs, several factors still limit its extensive use in clinical, one of which is enough titers of serum neutralizing antibodies. the development of mabs targeting the s protein of covs is regarded as a remedial strategy (table 4 ). potent mabs against s protein of human covs can be attained via transgenic mice immunization, convalescent b cells immortalization, and cloning of small chain variable regions from naïve and convalescent patients. 366 the majority of mabs interact with the rbd of s protein leading to the interruption of rbd-receptor binding and block the viral attachment. a few mabs react with other regions of s protein besides the rbd. 366 although binding to different epitopes, these mabs exhibit capacity to reduce the viral titers. two rbd-specific neutralizing mabs, mers-4 and mers-27, which were derived from single-chain variable regions, revealed suppressive effects against both mers-cov and pseudotyped mers-cov infection at nanomolar concentrations and were recommended as promising candidates for therapeutic interventions to mers. 232 based on similar mechanisms, other human mabs for mers-cov were also capable of competing with dpp4 for rbd binding and neutralizing the virus. 233, 234, [367] [368] [369] [370] when administrated to individuals at risk, some of the mabs were capable of preventing viral replication and contributing to block the transmission of mers-cov among human. 368 thus, such antibodies could be served as prophylactic strategies in clinical and valuable tools to guild the development of effective anti-covs vaccines. 234,368 from sars-cov to sars-cov-2, the emergence of severe human covs have taught us many lessons about the importance of rapid diagnostics and effective vaccines to control the outbreak caused by these viruses. due to the persistence of zoonotic sources in endemic areas, lethal covs remain existing in human society and may lead to the epidemic at any time. thus, a priority is to develop vaccines targeting conserved alleles and providing broad-spectrum protection against varied viral strains. since the emergence of sars-cov and mers-cov, several strategies were applicated in vaccine design, including inactivated virus vaccines, live-attenuated virus vaccines, viral vector vaccines, nanoparticles, recombinant protein subunits vaccines, and dna vaccines (table 4) . 371, 372 and clinical trials have also been developed to test the efficacy of the novel vaccines (table 5) . effective vaccines are pivotal in blocking the virus spread from animals' reservoirs to human hosts. inactivated virus vaccines, preserving the viral structure and antigenicity but eliminating the infectious ability, could elicit neutralizing antibodies in animal models of sars-cov and show protection against viral replication when administrated with or without adjuvants. 372 which may be related to the disseminated infection observed in immunocompromised patients. in addition, live-attenuated virus vaccines can induce an innate and adaptive immune response and the protective value can last for a long time. 371 besides, other strategies for vaccines development are also evaluated in animal models. based on the experience of sars-cov and mers-cov, the development of novel sars-cov-2 vaccine is currently underway and requires more research. however, several concerns should be addressed about the vaccination. the first is the disease deterioration caused by vaccination. although this situation only appears in a small subset of sars vaccine studies, it is still a significant problem that needs to be properly solved. 372 second, the variability of s protein can mediate covs escape from neutralization, suggesting that recombinant protein subunits vaccines based on s protein may demand multivalent approaches. 376 last but not least, how to define ta b l e 5 important clinical trials with vaccines for sars-cov, mers-cov, and sars-cov-2 the emergence and prevalence of highly pathogenic cov severely threaten public health. a task of top priority is to make clear the viral structural and epidemiological characteristics and block the viral dissemination as well as the progression of the disease, at the first case. to date, further understanding of the life cycle and the pathogenesis of emerging human covs makes current therapeutic strategies of antiviral infection more rational. repurposing existing antiviral drugs is an effective short-term strategy to deal with emerging cov such as the ongoing sars-cov-2. various agents with different targets have been evaluated in vitro and in vivo. but not all antiviral agents are capable of achieving better efficacy than in vitro, and in vivo studies are needed to select optimal agents. suitable animal models are particularly significant. however, there are only a few effective animal models available for the studies of covs treatment, which may postpone the clinical evaluation of drugs. besides, due to the diversity of viruses and the capacity of rapidly mutating, some antiviral reagents available for existing covs may become invalid. 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in the middle east date: 2016-11-25 journal: water, energy & food sustainability in the middle east doi: 10.1007/978-3-319-48920-9_17 sha: doc_id: 17731 cord_uid: xzfo5jjq food safety is a concern worldwide and according to the world health organization, developing countries are probably more at risk of foodborne illness because many of these, including those in the middle east, have limited disease surveillance and prevention and control strategies. specifically, the middle east and north africa (mena) region has the third highest estimated burden of foodborne diseases per population, after the african and south-east asia regions. however, it is difficult to determine what the burden is since little is published in peer-reviewed journals or government reports for public access. this chapter reviews 16 autonomous nations, namely, afghanistan, bahrain, egypt, iran, iraq, israel, palestine, kuwait, lebanon, oman, pakistan, qatar, saudi arabia (ksa), syrian arab republic (syria), united arab emirates (uae) and yemen. countries range in size from bahrain with 1.8 million inhabitants to pakistan with a population of 184 million. agriculture and local food production is much influenced by water availability for irrigation. water shortages are most severe in the gulf countries which rely on aquifers, desalination, and recycled waste water for most of their water supplies. this means that most food is imported which is expensive if not subsidized through petrodollars. this impacts food security which is a particular concern in countries under conflict, particularly, syria, yemen and iraq. gastrointestinal infections are frequent in this region from salmonella typhi and other salmonella spp., shigella spp., campylobacter jejuni and c. coli, rotavirus, hepatitis a virus, parasites, and more rarely from aeromonas, yersinia enterocolitica, brucella spp., and middle east respiratory syndrome coronavirus (mers-cov). reports indicate that children are the most susceptible and that many isolates are multidrug resistant. chemical contamination of water supplies and crops are probably more of a concern than published reports indicate, because of widespread indiscriminate use of fertilizers, antibiotics, and pesticides, coupled with increased industrial pollution affecting the water supplies. like many other parts of the developing world, foodborne disease surveillance is limited and outbreaks are most often reported through the press but with insufficient detail to determine the etiological agents and the factors contributing to the outbreaks, leading to speculation to the cause by those interested or responsible for food prevention and control. however, there are some well investigated outbreaks in the region that have those details, and reveal where the shortcomings of both the establishments and the inspection systems have been. where the causative agents are known, the kinds of pathogens are generally similar to those found in the west, e.g., salmonella, but many outbreaks seem to have short incubation periods that point to a toxin of some kind of chemical or biological origin, but these are almost never identified. because of sectarian warfare, residents and refugees have been given food that has made them sick and solders? have been deliberately poisoned. research has been focused on microbial contamination of locally-sold foodstuffs and manager and employee knowledge of food safety and hygienic conditions in food preparation establishments. an innovative pilot project in qatar is to use seawater and sunlight for raising crops through the sahara forest project. all countries have some kind of food establishment inspection system, but they tend to be punitive if faults are found in management or employees on the premises rather than being used for their education for improving food safety. restaurants may be closed down and owners and employees fined for often unspecified infringements. however, some food control agents are moving towards employee training through seminars and courses before problems occur, which is a good disease prevention strategy. unfortunately, many of the food handlers are from asian countries with languages other than arabic and english, which makes effective food safety communication and training difficult. tourists visiting popular resorts in turkey and egypt have suffered from foodborne illnesses, usually of unknown origin but poor hygienic conditions are blamed with law suits following, and the adverse publicity affects the long-term viability of some of these resorts. food exports, important for local economies, have occasionally been contaminated resulting in recalls and sometimes illnesses and deaths, notably fenugreek seeds from egypt (e. coli o104:h4), pomegranate arils from turkey (hepatitis a virus), and tahini from lebanon (salmonella). overall, in recent decades, the middle east has made strides towards improving food safety for both residents and foreign visitors or ex-pat workers. however, within the countries there are large discrepancies in the extent of effective public health oversight including food safety and food security. currently, almost all of the countries are involved to a greater or lesser extent in the civil wars in syria and yemen, or are affected through political tensions and strife in egypt, iraq, iran, israel, palestine, lebanon and turkey. in addition, the current overproduction of oil on a world-wide scale has led to a rapid decrease in revenues to most gulf states. all this points to a severe setback, and an uncertain foreseeable future for improvements in obtaining both sufficient and safe food for residents in this region. the world health organization (who) eastern mediterranean region, comprising countries in the middle east and north africa (mena), has the third highest estimated burden of foodborne diseases per population, after the african and south-east asia regions. according to the who ( 2015a), more than 100 million people living in this region are estimated to become ill with a foodborne disease every year and 32 million of those affected are children under 5 years. diarrheal diseases caused by e. coli, norovirus, campylobacter and nontyphoidal salmonella account for 70% of the burden of foodborne disease. an estimated 3000 people die each year from unsafe food, caused primarily by diarrheal diseases, typhoid fever, hepatitis a, and brucellosis. both typhoid fever and hepatitis a are contracted from food contaminated by the feces of an infected person and the source of brucellosis is typically unpasteurized milk or cheese from infected goats or sheep. half of the global cases of brucellosis are in people living in this region, with more than 195,000 people infected every year, causing fever, muscle pain or more severe arthritis, chronic fatigue, neurologic symptoms and depression. cholera, which after a short incubation period of 2-5 days causing severe diarrhea and dehydration, is returning to those countries with limited public health infrastructure caused by conflict, such as iraq (agence france-presse 2015). the list of countries covered by this chapter is similar to that of who but leaving out north african countries except egypt (which has territory in eastern asia) and adding turkey which is not always considered in the region because it is not arabic, but has interesting food safety data. therefore, the countries under review are afghanistan, bahrain, egypt, iran, iraq, israel and palestine, kuwait, lebanon, oman, pakistan, qatar, saudi arabia (ksa), syrian arab republic (syria), united arab emirates (uae) and yemen. gulf countries bahrain, kuwait, oman, qatar, ksa and the uae have similar social, political, economic, culture, religion, language and ancestry with several similarities in their food control systems and food safety programs (al-kandari and jukes 2009). a food and agriculture organization (fao) report covering international investments in agriculture in the near east (not identical to the countries chosen for this chapter, but many of the findings apply) states that this region is characterized by a mix of very different countries' resources and incomes (tanyeri-abur and elamin 2011). the wealth in the richer countries of the region is primarily dependent on oil revenues and the past economic growth has been closely linked to the oil market; about 43% of regional gross domestic product (gdp) is concentrated in the high income countries (qatar, kuwait, uae, saudi arabia, and bahrain) which are home to only11.8% of the population in the region, and many of these are expatriates working in these countries. the report indicates that food insecurity varies sharply in the region but overall the percent of the undernourished population does not exceed 57% in most countries of the region, except for sudan, mauritania, djibouti and yemen where the proportion of undernourished exceeds 25%; however, in 2016 these percentages will be totally out of date for countries like syria and iraq and in neighboring countries where refugees have reached because civil war and jihadi terrorist groups have put considerable stress on public health facilities and food availability. the countries in the region however, are largely similar when it comes to the challenges in achieving sustainable agriculture and food security. for most of these countries, the overwhelming concern is to secure adequate and stable supplies of food at the national level, making food security a concern for both rich and poor countries of the region (tanyeri-abur and elamin 2011). the three major problems affecting most of the countries are (i) limited water availability; (ii) population growth; and (iii) heavy dependence on food imports. water scarcity in particular, is the most critical development problem in the region and the single most important factor in limiting agricultural growth, and water availability has been declining steadily since the late 1950s. the region as a whole has 70% less availability of renewable water per person in 2003-2007 than in 1958-1962 . lack of water for irrigating crops but also for potable water supplies affects many of the countries, particularly in the gulf region. it is important to note that the wealthiest countries are also those with the highest water depletion record, namely, the uae and qatar. the unprecedented growth in investment in agriculture is in large part a result of the food crisis of 2007, which brought about a rethinking of agricultural support policies, mostly in countries of the gulf and particularly saudi arabia, which has invested heavily in the last 30 years in large-scale agricultural production using up valuable water resources. saudi arabia announced in january 2008 that it would phase out wheat and agricultural production in the course of the next 8 years. in july 2008, qatar and uae took similar policy decisions (tanyeri-abur and elamin 2011). crops grown in the region may serve as fresh food sources for the population, but much of the food is imported with limited locally processed products, and if the policies of ksa, qatar and uae expand to other countries, more will be imported in the future (tanyeri-abur and elamin 2011). thus, the main foodborne disease issues are with homemade, restaurant and street food, where isolated claims of illness are followed up by inspections and possible punitive action by public health agencies responsible for food safety. those countries that rely on tourism for their main source of gdp have sometimes been damaged by adverse publicity, e.g., egypt, and to a lesser extent, turkey and lebanon. according to the food and agriculture organization, less than 4% of the world agricultural trade is conducted in the region. even though by tradition many of these countries relied on growing their own food, today some of these countries import almost 90% of their food; 2.3% of the food in the world alone was imported to saudi arabia and united arab emirates in 2007, and the food trade balance in food in middle east is negative, estimated at over 50 us $ billion dollars (tajkarimi et al. 2013 ). there are specific restrictions prevalent in the arab-speaking countries related to islam and judaism with the prohibition of eating pork and blood, the drinking of alcohol, and mixing dairy foods and meat under halal and kosher food laws. therefore, parasites related to pigs, e.g., trichinella and taenia spp., are unlikely to be prevalent in these populations. however, there are many muslim and jewish feast occasions with large gatherings such as eid linked to ramadan and particularly the muslim hajj, which put a strain on food preparation, distribution and storage. good health conditions for travelers to saudi arabia for the pilgrimage to mecca (hajj) are critical and any incident that occurs has to be quickly contained to prevent extensive infectious disease outbreaks (memish and al rabeeah 2011) . traditional middle eastern foods are mainly related to legumes, leafy greens, fruit, dairy products and meat on special occasions; details can be found in brittin (2010) . in urban areas today, grocery stores and supermarkets can supply most of the food requirements of a family but imported foods tend to be expensive. also, some fruit and vegetable items are seasonal and are only available once or twice a year such as local plums, almonds and bananas, which tend to be cheaper than imported varieties. quality of raw produce in stores varies but they often have short shelf lives and can spoil quickly because of harvesting ripe products, bruising, and high storage temperatures. traditional rural foods include aromatic stews, stuffed vegetables, wild leaves, pulses and cracked wheat, and occasional goat or lamb meat. a typical middle eastern meal starts with a variety of cold and hot mezze (appetizers), salads and pastries, especially in greece, turkey and lebanon. many contain herbs, cheese, pickles, nuts, seeds, and parsley and lettuce are widely eaten in salads or traditional mezzes. most mezzes are vegetarian and fresh fruits and vegetables are an integral and important part of the cuisine when they are in season. tabbouleh, a salad where parsley is a major ingredient with small pieces of tomato, and some bulgur (ground wheat) in it, is often served in leaves of romaine lettuce or raw cabbage. almost as popular is fattoush, a mixed bowl of lettuce, tomatoes, cucumbers, and fried or toasted pita chips, typically seasoned with a dusting of sumac and pomegranate molasses. since leafy greens do not have a final decontamination step, they are at risk from environmental fecal contamination as reported in lebanon by faour-klingbeil et al. (2016) . hummus, a smooth chickpea paste made with tahini/tehineh, lemon juice or citric acid, garlic and salt, and often served with olive oil, is the most ubiquitous mezze. since tahini and hummus are major exported products from the region, particularly lebanon, they are prone to salmonella contamination, and are sometimes recalled from other countries, which is damaging to the local economies. dairy products are also served regularly at meals and these are locally made or imported. labneh, strained yogurt, very similar to greek yogurts, is widely used as a base for mezze which might have olive oil, pine nuts or za'atar (a mixture of thyme, sumac, and sesame seeds) added. cheeses including the popular haloumi are frequently served in restaurants. shawarma/ shwarma is frozen or refrigerated raw or marinated meat (lamb, beef or chicken) cooked on a vertical rotisserie popular throughout mena countries and now frequently seen in western nations. higher fish consumption tends to be close to where these are locally caught, either sea or river netted. one example from iraq is masquf (split large fish cooked on stakes over a fire, and eaten outdoors by a river, served with slices of tomato and onion and arab bread. crustaceans are less frequently eaten but can be obtained from imports. cosmopolitan foods are widely available in the larger cities, as are multinational fast-food chains. foodborne illnesses have been sporadically reported throughout the region over the past decades and global assessments of the kinds of problems encountered reviewed, e.g., todd (2001) and al-mazrou (2004) and more recently by tajkarimi et al. (2013) . these last authors indicate that reporting foodborne disease is functioning well in jordan, kuwait, oman, saudi arabia and uae, compared to other countries in the region. however, the foodborne outbreak surveillance systems in middle eastern developing countries are still limited with reporting of less than 1% of the actual outbreaks; one reason is that many foodborne illnesses occur in homes and those ill may not visit medical care facilities. in addition, available laboratory analytical support for public health agencies is often minimal or lacking, even though some research institutions may have up-to-date equipment and technical expertize. change is gradually coming and a food and drug authority has been established in both saudi arabia and jordan (al-kandari and jukes 2009). also, new food legislation has been initiated by egypt, lebanon and syria (tajkarimi et al. 2013) , but is currently stalled in last two countries. improvements in inspection service, hand held computers, customized software and improved surveillance systems are some examples of developments in food safety systems in the region. jordan, saudi arabia and bahrain have been developing unified food safety activities from farm to fork (al-kandari and jukes 2009). however, there is a need for substantive food safety education for all foodservice staff. increasing quality and quantity of the food safety training and human resources in governmental agencies in the region will improve the public health infrastructure. for example, the municipality of dubai has established an international annual food safety conference to improve the food safety education system of those in the region, now in its 10th year (2016). the following sections of the chapter focus on five aspects: gastrointestinal infections; foodborne disease outbreaks in specific countries; food safety related research and surveys; issues relating to tourism and exported food; and government oversight of the food industry, with specific examples from countries in the region. gastrointestinal diseases are frequently encountered in the middle east and many etiological agents have been identified where specific studies have been carried out to look for bacterial, viral and parasitic pathogens. the average annual incidence of culture-proven shigellosis in israel was 97/100,000 from 1998 to 2012, but each reported case was considered to represent 25 cases indicating the high burden of the disease in the country (cohen et al. 2014) . orthodox jewish communities, living in highly crowded conditions and with a high number of children aged <5 years were the epicenter of country-wide biennial propagated epidemics of s. sonnei shigellosis. s. flexneri was the leading shigella serogroup in israeli arabs. isolates showed high rates of resistance to ampicillin and trimethoprim/sulfamethoxazole, but very low rates to quinolones and third-generation cephalosporins. there is no indication if foods or water were vehicles of these shigellosis cases. also, in israel a study of pregnancy-related listeriosis cases from 1998 to 2007, identified 166 cases, resulting in a yearly incidence of 5-25 cases per 100,000 births (elinav et al. 2014 ). there were 29 fetal deaths, two neonate deaths and one maternal mortality. the incidence of israeli pregnancy-associated listeriosis has a high yearly variability and is one of the highest worldwide. the geographical distribution varied greatly between years and had a different epidemiological pattern compared with nonpregnancy-related listeriosis. the sources of the infections were not studied but all listeriosis cases have a foodborne link. this has to be further researched as to diet, and the unawareness of the israeli public of the risk for certain food products contributing to the extremely high incidence in israel, in both general and pregnancy-associated listeriosis, as occurs in other countries. a total of 132 stool samples were collected from palestinian patients with acute diarrhea from which 12 (9.1%) yielded enteropathogenic bacteria. salmonella, campylobacter coli/ jejuni, and aeromonas hydrophilia were isolated in equal numbers from samples 3/12 (25% each), shigella boydii 2/12 (16.7%), yersinia enterocolytica 1/12 (8.3%) (abdelateef 2011) . many strains were antibiotic-resistant. children younger than 5 years old were more susceptible to infectious diarrhea; in addition, diarrhea was more frequent in those living in crowded houses, and in houses rearing poultry, including pigeons. salmonella enterica serovar typhi continues to be an important public health problem in kuwait. analysis of the isolates from 163 patients, collected between 1995 and 2003, showed that the majority were from patients from the indian sub-continent, and many strains were drug resistant (dashti et al. 2008) . typhoid fever in kuwait is predominantly associated with those who have traveled from endemic areas to work in kuwait. the circulation of enteric viruses among the population of cairo, egypt, between march 2006 and february 2007 was studied by kamel et al. (2009) . at least one type of virus was detected in 50% of fecal samples, 57.4% of which were positive for rotavirus, 26% for norovirus, 10.4% for adenovirus, and 1.7% for astrovirus. over 10% of infections were mixed infections. among the noroviruses, half belonged to the predominant ggii.4 cluster which were similar to those circulating elsewhere, but there were also new ggii.4 variants that were not associated with any previously known ggii.4 isolate. although norovirus is rarely implicated in foodborne outbreaks compared with the us and other western countries, it is clearly present in egypt. further studies are required to assess the disease burden of enteric viruses in egypt and the impact of atypical strains. the disease burden of hepatitis a and e in egypt is one of the heaviest worldwide, based on serological analysis, with hav infections occurring very early in life, with almost 100% seropositivity after the first years of life (kamel et al. 2011) . to determine the actual contamination levels in the environment, these authors conducted a survey of hav and hepatitis e virus (hev) in sewage in cairo. hav was detected by rt-pcr in 11 of 76 (15%) sewage samples. in addition, all the hav-positive samples were also positive for enteroviruses. that only one stool sample was hev-positive might be explained by the lower level of excretion of the virus in stools, the fragility of the virion in the environment, and technical difficulties in concentrating and amplifying the virus with standard methods. bacterial etiology was found in 15.2% of cases of childhood diarrhea in dhahira, oman, mostly shigella sonnei and to a lesser extent salmonella (patel et al. 2008) . antibiotics were prescribed in 36.2% of cases and the resistance to the common antibiotics tested was low. one reason for the low pathogen isolation rate could be that many cases had viral etiology. rotavirus was detected in stool specimens from 1712 (49%) of 3470 children, who were admitted to 11 regional public hospitals in oman for a median of 3 days with severe diarrhea (al awaidy et al. 2009) . a diverse rotavirus strain pattern in oman was identified with g2 (37%), g1 (38%), and g9 (11%) accounting for most of typeable strains. the authors estimated the burden for the omani government at us$791,817 and us $1.8 million annually to treat rotavirus-associated diarrhea in the outpatient and hospital settings, respectively. they recommended a rotavirus vaccination program that would substantially reduce the burden of severe diarrhea among children in the country. unlike the above countries where the health care system functions for most residents, though not always to western standards, the same cannot be said for pakistan, particularly in rural areas. poor nutrition combined with diarrheal and other foodborne diseases puts the population at risk for serious illness and death, especially among infant and children in pakistan (akhtar 2015) . cholera, campylobacteriosis, e. coli gastroenteritis, salmonellosis, shigellosis, typhoid, and brucellosis have been demonstrated to be the major foodborne illnesses in the country as well as infectious diseases caused by viral and parasitic agents. many fatalities have been associated with food poisoning but the actual agent has rarely been determined. many health experts believe that rapid spread of gastrointestinal diseases cannot be controlled if the public has no awareness of prevention and control measures against cholera and other forms of gastroenteritis, and that in most parts of the country, sewage is continuously contaminating streams, lakes, springs, wells, and other drinking water sources (qasim 2009 ). in may 2011, an epidemic of diarrhea and gastroenteritis occurred in kamalia, toba tek singh, with over 30 children and others being admitted to hospitals which had few medical supplies. apart from lack of potable drinking water, the main reason given for the rise in cases was the heat of summer when there were frequent power cuts so that food "rots" or becomes "stale" (islam 2011) . in remote areas of pakistan, cholera has been responsible for many outbreaks. two examples in july and august of 2013, both in areas of conflict near afghanistan, give an idea of local but severe outbreaks. in one case authorities seemed not to want to be involved and in the other vaccinations are carried out. although water is the primary vehicle of the vibrio cholerae pathogen, it can easily contaminate prepared foods through poor hygienic practices. in july 2013, five deaths from cholera occurred in pashtoon kot area, balochistan region of pakistan (federally administered tribal areas) along the afghan border (staff 2013), some 70 km from quetta, in the absence of any emergency medical aid. the condition of an additional 20 people suffering from the disease was said to be critical. a local tribal elder expressed the fear that outbreak of cholera might cause loss of life at large scale. he complained that the doctor and paramedics deployed at the basic health center in panjpai live in quetta and are rarely seen at the center. officials of the provincial health department appeared to be unaware about the cholera outbreak and loss of lives (or ignored these), as they sent no medical teams to the affected area. in fact, pakistani government rebuffed international media's claims, and did not respond to requests to dispatch healthcare professionals to the balochistan area. it was assumed the outbreak would continue without medical aid. in 2012, cholera outbreaks killed hundreds of people, mostly children, in flood-hit districts of nasirabad, jaffarabad and jhal magsi where waterborne diseases were reported at a large scale because of consumption of contaminated water by local people. in august 2013, two people died and 320 others had fallen ill, following a cholera outbreak in kurram tribal agency near afghanistan (hussain 2013) . dhand and kudiad khel were the worsthit areas but vaccinations were carried out amid tight security, and tribesmen were instructed not to drink water directly from the well and boil it first instead since the wells had been contaminated from the rain water. around 100 people were shifted to parachinar headquarters hospital, while others were discharged after medical aid. sometimes diseases kept at bay by functioning public health systems come back when these break down as is occurring in a few of the countries embroiled in internal strife and outside attacks. for instance, in iraq in october, 2015, >1800 cases and 6 deaths of cholera occurred which started along the euphrates valley in september with the governorates of baghdad and babil, south of the capital, being the worst affected with more than 500 cases each. the epidemic then spread to the northern autonomous kurdish region, which hosts hundreds of thousands of people displaced by conflict from other parts of iraq (agence france-presse 2015). a previous outbreak killed four people in the kurdistan region in 2012. the united nations says the number of people displaced by conflict in iraq since the start of 2014 has topped 3.2 million which would exacerbate the spread of the disease. authorities blamed the cholera outbreak mostly on the poor quality of water caused by the low level of the euphrates. limited vaccination programs are in place in areas of conflict. in october, 2015, two persons arriving in kuwait from iraq tested positive for cholera and both were provided proper treatment and recovered. the ministry of health recognized that further cases could be discovered among people arriving from iraq, but because kuwait has a well-structured health infrastructure with water and sewers grids, and a supply of healthy and safe food, the disease should not spread into the kuwaiti population (anonymous 2015a) . probably there are some cases in yemen and syria, countries also with limited public health infrastructures, but have yet to be identified. in saudi arabia, a country with a well-maintained health system, the main infectious disease concern today are the infections and deaths arising from exposure to the middle east respiratory syndrome corona virus (mers-cov), which has reservoirs in camels and bats (todd and greig 2015) . a potential food source for this virus and other pathogens is from unpasteurized camel milk, as camel farmers drink the milk as well as being exposed through other aspects of camel contact. this brief review indicates that diarrheal diseases, caused by cholera, dysentery, hepatitis a, salmonellosis, shigellosis, typhoid fever, and other enteric diseases through water and food are major contributors to ill health in the region in agreement with the who (2015b) report on global estimates of foodborne diseases. in the region, not very many outbreaks of foodborne disease tend to be investigated, or at least reported publically, and those that are tend to have fatalities or are very large. for instance, in june, 2009, two children and one adult were brought to a hospital in dubai, uae, with suspected food poisoning (vomiting) after they ate take-away food (the father was out of town). although the mother eventually recovered, the two young children (5 and 7 years old) died, one on arrival and the other the next day. the cause was not determined (saberi and scott 2009) . it is not known if the family or restaurant was primarily responsible for the deadly gastrointestinal attack as bacteria can multiply quickly in the hot summer months, and the public had been recently warned to minimize eating out at this time of year, especially at smaller eateries where hygiene levels are often of lower standard. a toxin was likely involved to cause fatalities so rapidly, but it could have been an accidental contamination of the food with a chemical such as a pesticide, as much as it could have been with an enterotoxin produced by staphylococcus aureus or bacillus cereus through careless ambient temperature storage. unfortunately, this was one episode in a string of incidents, most of them with fatalities, in the county. in april, 2007, a 3-year-old died of suspected food poisoning in sharjah, and in august, a 10-year-old girl died of food poisoning in abu dhabi. in march, 2008, six people fell ill after eating buffet food at a restaurant in the large ibn battuta mall, dubai; in november of the same year, 14 employees at a cement factory were hospitalized after consuming what was considered rotten food prepared at the factory kitchen in another emirate, ras al khaimah. in may, 2009, a 4-year-old girl died of suspected food poisoning in sharjah. the indian family of four rushed to the hospital after series of vomiting but were too late to save the girl. dubai has been reporting foodborne outbreaks and cases through its foodborne disease investigation and surveillance system since 2011; in that year there were 1663 cases reported in the first nine months (saseendran 2014) . in 2013, 1123 suspected cases of foodborne illnesses were reported but only 518 cases were confirmed. no deaths were reported since the surveillance system was in place. egypt has had a particular problem with foodborne illnesses in universities and schools, mostly without a confirmed etiology, which seem to be related to poor food quality. food poisoning is not uncommon in egyptian university dormitories, where basic hygiene standards are often not observed, but the following outbreak was one of the largest. on april 2, 2013 hundreds of egyptian students angered by a mass outbreak of food poisoning at a cairo university stormed the offices of the country's top muslim cleric and university president, ahmed el-tayeb, because of the 479 students who were hospitalized after a meal served at the university dormitories in the nasr city district of cairo (associated press 2013). the university is affiliated with al-azhar mosque, the world's foremost seat of sunni muslim learning, and awards degrees in sciences and humanities, as well as in religious studies. in the protest, thousands of al-azhar students blocked roads, broke into el-tayeb's offices by the main campus, and chanted slogans against the university's management. the causative agent was unknown, and only with the incubation period, types of symptoms and their duration would it be possible to consider the potential etiologies of this illness. because of their poor quality, campus meals were not very popular before they were being blamed for the current food poisoning outbreak. although investigators were not able to find a specific cause, the university suspended its food services director and some other staff members. within a few weeks food poisoning affected 161 students on april 29, 2013 at the same university, al-azhar (masriya 2013) . investigations were initiated within the university and by the ministry of health, and apparently "bad tuna" had been served at the campus cafeteria; no further details were given. if tuna was the vehicle of the outbreak, scombroid poisoning was the likely cause of the illnesses. the allergic-like symptoms generally begin 15-60 minutes after ingestion and usually resolve in a few hours. scombroid fish poisoning occurs after fish, most frequently tuna, with high levels of accumulated histamine or other biogenic amines, is eaten. but "bad tuna' could equally be contaminated with bacterial or viral enteric pathogens with a longer incubation period. a month later there was another outbreak. because at least three outbreaks of food poisoning occurred at al-azhar university between april and may 2013 with over 700 cases of food poisoning detected in the university's male dorms, the dorm's director, the university's kitchen manager and eight chefs were sentenced in november, 2013, to 5 years in prison with a financial bail. in a similar situation, egypt's top prosecutor ordered a swift investigation into the 178 cases of food poisoning reported in two primary schools in october, 2014, in suez (masriya 2014 ). an official of the ministry of education indicated that the poisoning was caused by the consumption of milk provided by the schools. the distribution of milk to all schools in the governorate was halted until the milk's validity was ensured. if milk was responsible, the etiological agent could be bacillus cereus enterotoxin if the onset time was short, or less likely an infectious disease pathogen such as salmonella or e. coli o157:h7. on january 1, 2015, 150 female students were diagnosed with food poisoning at al-azhar university in upper egypt's assiut/ asyut governorate, by the banks of the nile, and were briefly hospitalized in an assiut city (anonymous 2015b) . this follows a similar incident which occurred in april 2014 when 29 students, also in the girls' dormitories, contracted food poisoning on the university campus in luxor. this report also flags two major poisoning incidents involving at least 500 students ill consecutively at its campuses in cairo in 2013 (probably the ones already discussed). the reason given for these repeated mass foodborne illnesses among university students is the quality of the food served them. apparently cheap, subsidized food is poorly stored, cooked and distributed to the poorer university students. in most cases the attorney general would open a criminal investigation that would be closed without knowing the microbiological cause of these outbreaks. the promed-mena editor speculated that enterotoxins of staphylococcus aureus were the most probable cause of such communal food poisoning, as a toxic dose of less than 1.0 microgram in contaminated food is sufficient to produce symptoms of staphylococcal intoxication. this toxin level is reached when s. aureus populations exceed 100,000/g, a condition likely to be present in these university kitchens because of intense pressure on them to feed a huge number of students in a short time, taking into consideration that most of these kitchens lack basic hygienic measures with regard to safe food handling. the editor also considered shigella, with its low infective dose (10-200 depending on the species) as another possible agent. however, the incubation period and symptoms of s. aureus intoxication and shigellosis or dysentery are quite different. pakistan is similar to egypt in that much of the country is rural but with very large cities with high populations (total population is 89 million in egypt and 184 million in pakistan, the most populous of all middle eastern countries). in september, 2010, more than 250 of the 1400 flood victims at a relief camp in bengali boys sindhi section school in ibrahim hyderi vomited after eating cooked food and then fell unconscious; 59 of them had to be taken to a nearby hospital (aligi 2010) . a local philanthropist had been providing cooked food to the flood victims but by the time the food arrived at the relief camp, the cooked rice had turned "stale". since the rice did not show any sign of spoilage, it was served to the flood victims. a similar incident had taken place 3 days earlier at another town where more than 80 flood affectees had fallen unconscious after consuming "stale" food and 10 were hospitalized. none was seriously affected. during the investigation, it was noticed that the sanitary situation in and around the relief camps was very poor. even though the reason for the illness was not determined, the police took action against the donor and two caterers. in fact, based on the information of the vehicle and the symptoms, bacillus cereus enterotoxin which is known to be produced in boiled rice, was the most likely agent. in the following two outbreaks yoghurt is blamed for the serious illness and deaths though details of the symptoms are not given. rapid onset of symptoms indicates the presence of a toxin of some kind, although yoghurt is not a food known to be frequently contaminated with pathogens because of its high acidity. either the yoghurt was made under very unhygienic condition with the source of the milk perhaps being spoiled (possibly containing bacillus cereus enterotoxin), or a chemical had been added accidently such as a pesticide, or deliberately and illegally to enhance the flavor. however, it is possible other foods were involved and yoghurt was not the contaminated vehicle. in january 2011, in lahore, a hospital employee died and two other employees became critically ill after eating contaminated yoghurt. the three employees ate rice with yoghurt at a local restaurant (ians 2011) . action was taken against the restaurant owner and manager. no further details are known. in early april, 2014, a rawalpindi family of ten became seriously ill after eating a home-prepared evening meal where yoghurt was suspected to have been the contaminated food, and they were taken to a hospital, where a teenage boy and 7-year-old girl died (asghar 2014) . the surviving family members remained in critical condition for some time but eventually recovered; the cause of the illnesses was not discovered, although it was postulated by a relative who had eaten the yogurt with the meal that it was possibly poisonous or, strangely he thought a lizard might have fallen into it. in february 2012, at least four people died and another seven were hospitalized in a critical state after eating home-cooked biryani (a dish made with spices, rice and meat or vegetables) in a suburb of karachi (mahmood 2012) . the owner of a grocery shop, who provided the ingredients, was arrested, and a sample taken for analysis. it is not known if any toxin was found. a month later in march, in faisalabad, more than 60 children and women were ill after eating contaminated aalo-chanay (potatoes, chick peas, onions, tomatoes and spices) purchased from an unidentified vender (anonymous 2012c) . as soon as the children ate the aalochaney, they felt ill and started vomiting. although they were immediately rushed to a rural health center, one boy died. a medical opinion was given that the eaters suffered from "diarrhea and cholera". however, the onset was too rapid for anything but a toxin of some kind, most likely heat-resistant since the aalo-chanay was cooked. also, in march 2012, as many as 47 student nurses and eight staff nurses were hospitalized with acute food poisoning at a hospital in rawalpindi after eating food at the nursing hostel, but none was critically ill (anonymous 2012d). the nurses residing in the hostel started reporting complaints of vomiting and diarrhea along with high-grade fever at an undisclosed time after a meal. the hospital administration was criticized for failing to provide safe food and drinking water to its employees and demanded immediate inquiry into the case, but none was reported on. the illnesses are consistent with an enteric infection such as salmonella or norovirus. in april 2015, at least 20 constables suffered from diarrhea and were admitted to hospitals when they ate food during the sehat ka insaf program, which is a blanket method of administering the polio vaccine along with eight other vaccines, hygiene kits and vitamin a drops in order to circumvent polio-specific terrorist attacks in pakistan. local administration purchased packed food, including piece of chicken and juices from a local supporter (mayar 2015) . no further details are given but the chicken could have been undercooked or cross-contaminated with enteric pathogens such as salmonella and campylobacter; if the packs had been left at ambient temperatures for some time, these pathogens could have multiplied on the chicken to large numbers. over thirty children in faisalabad were hospitalized over 4 days because of diarrhea and gastroenteritis, three seriously, and other children were expected to be ill. undetermined contaminated food was postulated as the cause, more than usual because of the extreme seasonal heat combined with frequent power outages to allow rapid bacterial growth in contaminated food. the unavailability of clean drinking water was mentioned as a contributing factor to the increasing number of gastrointestinal disease cases. hospital administrators complained that vaccines and medications were required but were not forthcoming from the health department. probably many family meals were contaminated because of the lack of potable water and any unspecified enteric bacterial pathogens present could grow rapidly in the heat. children are more vulnerable than healthy adults to infections which might explain the high proportion of sick children seeking medical help. botulism outbreaks occur periodically in iran. in a study of stool and serum specimens of 115 patients with clinical symptoms of botulism, who were at inpatient and outpatient medical centers in tehran and other areas of iran, between april 1984 to august 1994, specimens of 73 patients showed the toxin and spores of c. botulinum (modarres 1997) . type e was the most common causative agent found in this study, being responsible for 71.2% in all specimens; other etiologic types, in order of frequency were types a (16.4%) and b (12.3%). type e strains are typically associated with fish and freshwater and marine sediments. the results of this study indicate that the cases had consumed salted fish, smoked fish and canned fish, along with cans of green beans and cucumbers. a similar result over a decade later confirms that c. botulinum type e is a major pathogen in iran. in gilan province, of 146 fish samples collected in 2008, 11% of processed fish and 7.5% of non-processed fish contained clostridium botulinum, mainly type e (tavakoli and imani fooladi 2011) . the processing is insufficient to kill the spores or reduce much of toxin produced because the fish tend to be partly cooked with the intestines kept intact. a total of 131 traditional food product samples (57 cheese, 11 kashk [a type of dried yoghurt or thick cream], and 63 salted fish) were examined using a bioassay method for detection of clostridium botulinum toxin (hosseini et al. 2010) . standard monovalent antitoxins were used to determine the toxin types. c. botulinum toxins were detected in 4.6% of examined samples (3.5% of cheese samples and 6.4% of salted fish samples). none was found in kashk samples. c. botulinum types a and e were dominant in cheese and salted fish samples, respectively. consumption of these traditional foods either raw or processed may contribute to foodborne toxicity in iranian populations. in may 2014, a quickthinking mother immediately brought her 7-month old boy to an israeli hospital when she saw he was suffering from vomiting, difficulty in breathing, listlessness, glassy-eyed, apathetic, and an inability to nurse or eat (bender 2014) . a doctor at the hospital diagnosed the child as suffering from infant botulism. he decided to treat the baby with the antitoxin stored in the emergency stocks, even before they got back the lab test results. the hospital like all israeli medical facilities keep ample supplies of biological and chemical warfare antidotes on hand in case of war or terrorist attacks, and staffers are regularly drilled in dealing with the symptoms of various chemical, neural and blister agents. the infant started recovering soon after the administration of the antidote. in the rare disease of infant botulism, spores of clostridium botulinum are ingested and the infant's flora is not mature enough to prevent germination and slow growth of the toxigenic pathogen. it is entirely possible that infant botulism occurs more frequently in the region but is not diagnosed. foodborne disease surveillance depends on an infrastructure of reporting and diagnosis in hospitals, epidemiologists, and food testing laboratories. lebanon is an example of a country where modernization in public health seems to occur at a glacial pace. however, diseases including those of foodborne and waterborne origin, are documented and published. the law of december 31, 1957 regarding communicable diseases in lebanon mandates all physicians, from private or public sectors, in hospitals or ambulatory services, to declare to the epidemiologic surveillance unit of the moph all diseases considered a risk to public health. the data available at the ministry of public health (moph) are compiled from different sources, and the declaration of cases remains irregular and insufficient (moph 2012) . in 2011, foodborne and waterborne diseases were the most frequently reported in lebanon at a rate of 0.4 â�° (total of 1468 cases), with the highest rate in the bekaa (0.7 â�°) and the lowest in the south (0.1 â�°). the most common infection was viral hepatitis a, which represented 30.5% of the total food and waterborne diseases with 448 cases. there were also 362 cases of typhoid (24.7%), 311 cases of food poisoning (unspecified, 21.2%), 186 cases of dysentery (12.7%), 34 cases of brucellosis (9.1%, 15 cases of parasitic worms (1.0%) and 12 cases of hydiatic cyst (0.8%). no cases of cholera and trichinosis were declared. hydiatic cyst (cystic echinococcosisis) caused by echinococcosis (typically e. granulosis) is acquired by contact with animal feces contaminated with tapeworm eggs. sources include contaminated food (meat), water, and animal fur. cysts containing tapeworm larvae may grow in the body for years before symptoms appear. when cysts become large, they may cause nausea, weakness, coughing, and belly or chest pain. occasionally, well-investigated outbreaks are published; the following two examples are from lebanon and neighboring jordan. in may 2004, 32 employees suffered from diarrhea, fever, and abdominal pains 5.5-22.5 h (mean, 10.3 h) after eating chicken noodles au gratin at a catered lunch served at a bank cafeteria (hanna et al. 2009 ). a few cases had systemic infections. salmonella enteritidis (se) was confirmed in stool and blood cultures within 48-72 h after hospital admission of the first cases, and also in leftovers of the suspect food. the same dish had been served at the bank in the past with no apparent health problems. preparation normally started in the evening prior to the day the dish is served. however, in this instance, some of the constituents had been prepared 3 days ahead, because the dish was to be served on a monday, immediately after the week-end closure. no salmonella was found in rectal and nasal mucosal swabs taken from all 18 kitchen workers, or in the tanker water supply (although it had high fecal coliform counts), but se was found in a frozen batch of the same raw chicken breast consignment that had been used for the chicken noodles. the batch of chicken came from a large producer of poultry and eggs in lebanon, who was advised of its potential involvement in a major foodborne outbreak. however, the investigators were refused access to the poultry-producing facility. it is highly likely that contaminated chicken carcasses had been, and would continue to be, shipped to many parts of lebanon. that the same se strain occurred in the patients, the raw chicken, and the leftover food was confirmed through random amplified polymorphic dna polymerase chain reaction (rapd-pcr). it would appear the 3-day delay in the chicken noodle preparation was significant in allowing the salmonella present in the ingredients not only to survive but probably to grow; undercooking, cross-contamination, inadequate storage and reheating all may have played a role in the outbreak, but no more information was available to determine which of these were the key factors in the outbreak. the bank management decided to sue the caterer and because they were aware of apparently inefficient way that public authorities were conducting the procedure, they took the initiative to call upon an independent investigative team to obtain solid evidence to win any court action. the caterers, concerned that they would be the only party blamed for the salmonella outbreak, had succeeded in concealing some raw and cooked items from destruction by the public health authorities, which was their normal practice after a complaint. these items were central to establishing contamination upstream from the caterer's kitchen. no action seems to have been taken against the poultry producer who was the source of the se, a pathogen that is invasive of flocks and difficult to eradicate. the authors complained about the obsolete lebanese laws dating back to the 1930s that still governed what should be done following a report of "food poisoning". public health officers are mandated to stop the spread by destroying allegedly contaminated food items and closing down incriminated facilities. hanna et al. (2009) stated that this kind of action is generally lauded by the public but does not help determine the cause to develop appropriate prevention and control strategies. they also complained that because no investigation is typically done, many non-implicated foods and ingredients are wastefully discarded. the jordanian example is over two decades old, but is worth noting in detail. in september 1989, a 183-case outbreak of salmonellosis occurred in a university hospital in amman after employees, patients and visitors ate in the cafeteria. the incubation period ranged from 16 to 72 h. symptoms included diarrhea (88%), fever (71%), abdominal pain (74%), dehydration (34%), and bloody stool (5%); 84 were hospitalized (khuri-bulos et al. 1994) . cultures of eight food items were negative, but stool culture on 90 of 180 patients and 11 of 61 kitchen employees yielded salmonella enteritidis (se) group d1. a cohort study revealed a foodspecific attack rate of 72% for the steak and potato meal and 18% for the rice and meat meal. stratified analysis of the steak and potato meal revealed that the potatoes were implicated most strongly. cultures were obtained from all kitchen employees, who showed no symptoms of illness, but 11 of 61 grew se group d1. one asymptomatic, culture-positive employee had prepared the mashed potatoes on september 23, 16 h before the first case presented at the hospital emergency with severe gastroenteritis symptoms. all of the food workers had negative stool cultures 3 months earlier. the potatoes were mashed by machine, but peeled after boiling and mixed with milk by hand, using a ladle but no gloves. two different batches, the first of which was served exclusively to hospitalized patients and the second to a few remaining patients and employees, were prepared and served within 30 to 60 minutes of preparation. from the epidemiological data it can be assumed that the infected handler fecally contaminated only the second batch of potatoes, thus sparing most of the highly susceptible inpatients from exposure. furthermore, while potatoes clearly were implicated, individuals who ate steak only had an elevated risk of being attacked. this probably was due to surface contamination of foods being served on the same plate. kitchen employees harboring salmonella were excluded from work until they had three negative stool cultures taken 1 week apart; it took 6 weeks for them to return to work. stool surveillance that was routinely carried out in the hospital was ineffective in detecting infected employees to prevent this outbreak and the investigators recommended that employees adhere to proper hygienic practices including thorough washing of hands, especially when preparing food. today, salmonella is only one of many of the pathogens that can be encountered in foodborne illness. one of the newer pathogens, well established in the west is norovirus (nov), which causes more cases of foodborne disease in the u.s. than any other agent (scallan et al. 2011 ). in may 2009, a significant increase in acute gastroenteritis (age) cases was noted in the american health clinic at incirlik air base (iab) in adana, turkey. this increased rate of age led to discussions with local turkish military public health authorities, which confirmed that the turkish military community and the residents of adana were also experiencing an anecdotal increase in age illnesses (ahmed et al. 2012 ). an epidemiologic investigation was launched to attempt to identify the cause and possible source of this age outbreak at iab from may to june with the peak incidence of cases during the week of may 31-june 6, with a total of 71 patients seeking medical care at the clinic. of the total 187 infected persons, 82 patients completed the case survey, 79% reported diarrhea, 46% reported vomiting, and 29% reported fever. the median number of days between symptom onset and clinic visit was 2 days. during the 7 days prior to symptoms, 73% of respondents reported travelling off base, 56% reported eating off base, and 24% reported using an outdoor pool. this outbreak had a significant negative operational impact, degrading mission readiness with nearly 20% of the american population in a 1-month period affected. initiation of a clinic case-based investigation yielded 37 stool specimens in which nov was detected in 43%, with 81% of the positive nov specimens identified without a copathogen. dna sequencing data demonstrated that several relatively rare genotypes of nov contributed to this outbreak; four different genotypes were isolated from 16 positive specimens. two of the nov strains were previously reported in iraq and only from deployed troops, while the other two genotypes were reported in south africa and in the us. in turkey, little systematic data on circulating nov genotypes exist. however, giib/gii.4 strains have been frequently identified in turkish children with gastroenteritis; strains belonging to this genotype have been found in europe and mainly in children. previous reports from british troops deployed to iraq indicated that two nov strains isolated were responsible for cases of gastroenteritis there. similar mixed nov outbreaks have been previously observed and are often attributed to systematic failure of cooking/cleaning/drinking water supplies (ahmed et al. 2012) . one limitation of this investigation was that the survey was not used to capture data from a control group, those without recent age, preventing carrying out a risk factor analysis. another limitation was the lack of environmental samples that could be tested for nov in order to track the source of outbreak. from anecdotal information, it is likely many in the local population and the turkish military base were ill, but a formal outbreak investigation in the turkish population was never performed. from the multiple genetic types involved, one specific contaminated food or water source seems unlikely. the largest turkish nov outbreak was in keã§iborlu province of isparta county between april 5 and 17, 2010, with 1428 patients seeking medical help from the healthcare centers, after suffering from nausea, vomiting and abdominal pain (more frequent than diarrhea) (sâ�¬ ozen et al. 2014) . because of underreporting, the number of affected people was estimated to be higher. municipal water was the suspected source but no samples tested positive. as a cautionary note, the authors suggest that nov may not be the only causative agent of gastroenteritis outbreaks, especially from an undetermined fecal source, and bacterial, viral and parasitic agents should be examined together with the nov. in saudi arabia, a national policy for reporting, notifying, and recording incidents of bacterial food poisoning was established in 1984 (al-joudy et al. 2010). since then salmonella food poisoning outbreaks have been reported from different regions of ksa, exhibiting seasonal and regional variations, with chicken, meat, and rice being commonly incriminated food items, and frequently reported in the saudi epidemiological bulletin. al-mazrou (2004) reviewed the history of foodborne outbreaks in ksa and saw an increase over the last few decades, especially those caused by salmonella, with the main food vehicles being chicken, meat and eggs, and s. enteritidis being the most frequent salmonella serovar responsible. according to promed editorials, restaurants and communal feasts and institutional feeding (such as in school cafeterias, hospitals, nursing homes, prisons, etc.) where large quantities of food are prepared several hours before serving are the most common settings in which foodborne illness incidents occur (http://www.promedmail.org). for instance, in 2005, a hospital in the jizan region received 19 suspected food poisoning cases that were ill after taking meals from a restaurant, including a woman who suffered from severe diarrhea, abdominal pain, vomiting and dizziness (fagbo 2005) . the restaurant was closed down and three of its workers were detained pending the results of laboratory tests. the report of an investigative committee could not find a specific cause, but noted that the restaurant had earlier been responsible for some hygienic violations. in 2015, 80 cases suspected of foodborne illness after eating a meal at a restaurant were admitted to various hospitals in the najran region (alhayat 2015) . most of the cases were not seriously ill. no report was given on the samples that were taken from the suspected restaurant, which was closed temporarily. there is an interesting observation related to variant creutzfeldt-jakob disease (vcjd); four cases have occurred in the us since the disease was first diagnosed in the united kingdom in 1996 linked to consumption of cow meat suffering from bovine spongiform encephalopathy (bse); two of these were associated with the united kingdom (where bse was first reported), but one came from saudi arabia and the most recent case in 2014 had extensive travel to the middle east and europe (cdc 2014) . this may indicate some source of vcjd in the middle east including saudi arabia. one of the big concerns for ksa is the annual hajj with millions of muslims from around the world converging on mecca, in saudi arabia, each year. no other mass gathering can compare with the hajj, either in scale or in regularity, and various communicable disease outbreaks of various infectious diseases have been reported repeatedly, during and following the hajj (memish 2010) . in 2006, an outbreak during the hajj occurred where all the cases came from one tent occupied by 50 soldiers located in a government camp in mina, makkah province, near mecca (al-joudi 2007). the camp was served by a catering company that prepared and distributed three meals daily (breakfast, lunch, and dinner). a case was defined as any individual who developed diarrhea with or without abdominal pain after eating at the camp in mina in january, 2006. of the soldiers who were interviewed, 16 (39%) had developed gastroenteritis, most commonly manifested by diarrhea (100%), and abdominal pains (87.5%). the mean incubation period was 12.6 ae 4.9 h and the epidemic curve suggested a common point source outbreak. out of three served meals, lunch with a rice dish was found to have a statistically significant association with illness. unfortunately, no food remnants were found for sampling, and the results of stool cultures of all diarrhea patients, and rectal swabs from all food handlers were inconclusive. temperature abuse was cited as a contributory factor in this outbreak. based on the incubation period and symptomatology, bacillus cereus would be the most likely etiological agent. another example of a foodborne illnesses associated with the hajj occurred in 2011 when 81 bangladeshi pilgrims were taken to hospitals in madina (medina) after eating a meal prepared by an unlicensed caterer (promed-mena 2011). they suffered from abdominal pains associated with diarrhea and vomiting. the pilgrims were all treated and discharged, except for one who remained hospitalized. samples of the food they had eaten were sent for analysis but the results are not known. considering the mass of people converging on this small part of the middle east, it is surprising there are not more foodborne disease outbreaks. this may mean excellent food control by the authorities or some illnesses are simply not recognized and reported. at least 750 bahrainis suffered from food poisoning after eating catered sandwiches served during a wedding celebration, the biggest mass poisoning outbreak in the country's history (promed-mena 2002) . the wedding took place in the safala village, near the eastern island of sitra. all eventually recovered after treatment but one man who had sickle cell disease, died. teams were formed to investigate the outbreak, and blood specimens from all workers at the bakery who prepared the egg, cheese, and mayonnaise sandwiches along with leftover sandwiches and their ingredients on the caterer's premises were sent for bacteriological analysis. the bakery which supplied the sandwiches was closed by the public health directorate at the ministry pending the investigation's results. unfortunately, no final report was released to the public. the promed editor considered the etiological agent could be salmonella or staphylococcus aureus enterotoxin, depending on the length of the unstated incubation period. the region experiences some unusual type of illnesses relating to on-going hostilities. for instance, the united nations has been sending aid to reach besieged towns in syria, close to the lebanese border, but in october, 2015, it sent hundreds of boxes of "moldy" high-energy biscuits past their 'sell-by' date in september (320 of the 650 boxes transported) to zabadani and madaya, apparently causing food poisoning (afanasieva et al. 2015; muhkalalati and kieke 2015) . officials stated these could be the only cause of an outbreak of food poisoning among almost 200 residents who came to makeshift hospitals, mainly children who had vomiting, diarrhea and abdominal swelling almost immediately after eating the biscuits. the biscuits were described as "moldy and rotten and had been poorly stored". apparently, when the last aid order that was sent was filled, there was a shortage of food. the red crescent, who was filling the order, took some of the expired goods to complete it. however, these biscuits had only just expired and normally would not have posed any health risks to those eating them. nevertheless, the words poorly stored suggest that moisture may have encouraged microbial growth (visible mold more likely than bacteria because fungi can grow aerobically in the presence of the presumably elevated sugar content in the high energy biscuits). also, contributing to the symptoms, the residents of zabadani and madaya had been blockaded for 120 consecutive days, and their immune systems were extremely weak. refugees are also at risk of gastrointestinal diseases from contaminated water or food. up to two million syrian migrants fleeing syria due to the civil war were living in turkey, and supplying them with safe and secure food supplies is a challenge for any host country. one incident, no doubt, one among many indicates the risk of contaminated food. in april, 2015, five security forces were injured after syrian migrants in a tent city in turkey's southeastern province of mardin reportedly attacked guards over allegedly being poisoned from the lunch at the camp (anadolu agency 2015). some 17 syrian migrants were detained after the incident; 66 syrian migrants out of the 5230 currently residing in the temporary sheltering center in mardin's derik district applied to the center's hospital with symptoms of food poisoning, dizziness, and vomiting. after treatment they were discharged, none of them in a critical condition. although an investigation was conducted and samples from the lunch sent to the lab for analysis, no further information was available on the outbreak. promed speculated that if the lunch food was the vehicle, it would be a short incubation illness likely caused by staphylococcus aureus, bacillus cereus, clostridium perfringens, or possibly a non-biological toxin. these illnesses may cause vomiting, diarrhea, or both, and are usually short in duration (less than 24 h), and are not associated with prominent fever. in iraq, no recent foodborne disease outbreaks have been published, but no doubt many have occurred in the last decades with so much public health infrastructure dismantled. only the most newsworthy of outbreaks are being covered by the press today. iraq and other middle eastern countries are in sectarian turmoil and on two occasions islamic state (isis/is/isil) fighters (jihadis) were likely poisoned by cooks who infiltrated their camps. in november, 2014, a group of defected syrian soldiers (free syrian army men) who posed as cooks reportedly poisoned isis militants after they ate a contaminated lunch at the fath el-sahel camp, where 1200 of them were based (gee 2014 ). apparently about a dozen of the jihadis were killed and 15 taken to nearby field hospitals. the 'cooks' immediately fled, along with their families, with the help of fellow revolutionaries. seven months later, in july 2015, 45 jihadis died after ingesting an iftar meal eaten by 145 isis militants (akbar 2015; variyar 2015) . it remains unclear whether the jihadis, who were breaking their ramadan fast in mosul, iraq, died of accidental food poisoning or intentional poisoning, but it is likely a repeat attack of the earlier incident described above. the nature of the poison or details of the illnesses in either episode are not known. however, in both episodes, onset and severity of the attack were rapid, probably caused by a relatively tasteless chemical in lethal doses added to one or more foods. targeting the military by any means including poisoning food has always been a strategy of opposing forces. in february 2012, a deliberate attack was foiled when afghan border police detected a significant amount of bleach in fruit and coffee stored at their main border checkpoint between afghanistan and pakistan, a likely attempt to poison the afghan security forces (tucker 2012) . the police decided that although none of this food had been consumed, the level of contamination was high enough to cause serious injury, and it must have been done intentionally. there had been previous incidents of intentional food poisoning aimed at afghanistan's civil defense forces, including an episode in kabul in 2011 when several people were sickened. in 2012, in southern helmand province militants killed four afghan policemen and two civilians inside a police checkpoint by poisoning their yoghurt coordinated with an attack (anonymous 2012a). there had been several recent poisoning incidents involving members of the afghan national police, as part of attempts by the taliban to infiltrate the security forces; three police officers were reported missing, along with their weapons and a police vehicle, following that attack in helmand province. taliban militants had first poisoned the police officers' yoghurt before launching a full scale attack on the checkpoint. similar tactics had been used by insurgents in helmand before. the same thing happened again in january 2016 when a rogue policeman collaborating with insurgents in southern province of uruzgan shot dead 10 colleagues after first poisoning their food, but no further details are given (reuters 2016 ). turkey does have food laws that are supposed to limit food contamination and resultant foodborne illnesses. the 2000 turkish food code stipulates that all turkish food businesses have to provide food hygiene training commensurate with the work activities of their staff. to see what progress had been made in this area baå� et al. (2006) evaluated knowledge, attitudes, and practices concerning food safety issues among food handlers in ankara, conducting face to face interviews and administrating questionnaires. the majority of the 764 food handlers who responded (47.8%) had not taken a basic food safety training (and probably most of non-respondents had not either). the mean food safety knowledge score was 43.4 ae 16.3 of 100 possible points. the self-reported hygienic practices showed that only 9.6% of those who were involved in touching or distributing unwrapped foods always used protective gloves during their working activity. of those food handlers who used gloves, only 8.1% and 3.8% always washed their hands before putting them on and after removing them, respectively. in addition, there was a difference handlers' scores depending on where they worked. scores were higher for food handlers in catering establishments (50.4 ae 9.4), school food services (52.5 ae 9.2) and hospital food services (50.9 ae 9.2) than restaurants (47.3 ae 8.6), hotels (47.4 ae 8.3), takeaways (44.1 ae 2.5) and kebab houses (37.9 ae 6.7). these scores may also be biased upwards since they were self-reported and not observed practices. the study demonstrated that food handlers in turkish food businesses often have lack of knowledge regarding the basic food hygiene, e.g., critical temperatures of hot or cold ready-to-eat foods, acceptable refrigerator temperature ranges, and cross-contamination. those who were trained scored better, and the authors stated there was an immediate need for education and increasing awareness among food handlers regarding safe food handling practices. in istanbul from 2005/2006, thermophilic campylobacter was isolated from 11.1%, 21.6%, and 50.4% of beef, mutton, and chicken samples tested, respectively (bostan et al. 2009 ). there was no significant seasonal variation in the prevalence of the pathogen. c. jejuni was the species most commonly isolated from chicken meat, while c. coli was the most common in beef (63.3%) and mutton (63.9%) carcasses. campylobacter isolates were most often resistant to tetracycline (69.1%), followed by trimethoprim-sulfamethoxazole (64.2%), nalidixic acid (58.1%), erythromycin (56.9%), enrofloxacin (48.8%), ciprofloxacin (42.7%), chloramphenicol (36.2%), and gentamicin (26.0%). the results of this study suggest that a high proportion of meat samples, particularly chicken carcasses, are contaminated by campylobacters, most of which are antimicrobial-resistant strains. in yemen, the prevalence of salmonella in food was determined in sana'a city from april 2009 to april 2010 by ahmed (2013) . of the 362 different food samples collected from local markets, salmonella spp. were isolated from 26 (7.2%). the highest prevalences were in red meat (14.7%), chicken (12.1%), eggs (11.8%), cooked foods (9.5%), raw milk and milk products (5%), juices (4.8%), vegetables (4.4%), sandwiches (3%), and pastries (2.6%). serogroups identified were b, c1, c2-c3, d1, e1, and e4, and some foods contained more than one isolate with different serogroups, especially red meat. because handlers in foodservice facilities play a major role in transmission of foodborne diseases (greig et al. 2007) , studies have been carried out to demonstrate their knowledge of practices related to food safety. in jordan, osaili et al. (2013) measured food safety knowledge of food handlers working in fast food restaurants in the cities of amman and irbid. a total of 1084 food handlers in 297 fast food restaurants participated in this question survey study. the overall knowledge of food handlers on food safety concepts was considered to be fair (69.4%). the food safety aspect with the highest percentage of correct answers was "knowledge of symptoms of foodborne illnesses" (81.7%) and "personal hygiene" (79.9%), while the lowest percentage of correct answers was for "safe storage, thawing, cooking and reheating of the foods" (52.4%), critical practices to prevent the survival and growth of pathogens. the mean knowledge score of "personal hygiene" reported in the study was much higher than 51.5% and 31.8% reported by martins et al. (2012) and baå� et al. (2006) , for the food handlers in portugal and turkey, respectively. also, only 31.9% of respondents considered the duration of hand washing to be !20 s. when they were asked how they check that the poultry is sufficiently cooked, only 31% knew "when the meat has the correct thermometer reading", although 85% of the respondents had thermometers in their restaurants. about 25% of them answered that poultry is cooked "when it has been cooked for the stated time" (24%) and "when it looks cooked" (27%). about 50% of them would store leftovers on the steam table (40%) and in the refrigerator (53%) while about 27% of the correspondents would store leftovers at room temperature in kitchen or in the oven. a low percentage of the respondents (20%) reheated leftovers to the appropriate temperature (73 c). about 40% and 20% of the respondents had heard about salmonella and hepatitis a virus, respectively, but 10% of the respondents knew about listeria monocytogenes, staphylococcus aureus, bacillus cereus, escherichia coli o157:h7, clostridium perfringens, campylobacter jejuni, or shigella. food workers who had enrolled in a food safety training course had significantly higher total food safety knowledge score than those who did not take any training. there was no association between the experience or any other characteristic of food workers and total food safety knowledge score. this study suggests adopting proper food safety education training courses to food handlers, periodic evaluation of food handlers' knowledge and food safety training course materials. also, the authors considered that better pay for food handlers would improve the food safety status in foodservice institutions. similar concerns over practices that could lead to food contamination and foodborne illnesses were demonstrated in lebanon. a survey was conducted in beirut to evaluate the knowledge, attitudes and practices related to food safety issues of food handlers (n â¼ 80) in foodservice establishments (n â¼ 50), and to assess the influence of management type on enactment of safe practices on food premises (faour-klingbeil et al. 2015) . the data suggest that while respondents do have some knowledge of food safety aspects, substantial gaps in their knowledge and self-reported practices associated with critical temperature of foods and cross contamination remain, therefore posing health risks to consumer health. food handlers in corporate managed food outlets showed a significantly higher awareness on food safety practices. it is concluded that the management type is an integral element of the theory of planned behavior that influence food handlers' practices and substantiate the need for more research work on safe food handling in the context of food safety culture framework in food businesses. as in many other mena countries, there is a critical need for food safety education interventions and technical guidance fostered by synergistic participation of the private and public sector to support food handlers in smes (small and medium sized enterprises). parasites are not often looked for in middle eastern countries but they are frequent, and one of the ones of most concern for pregnant women is toxoplasma gondii which is transmitted through undercooked meat and cat feces. since stray cats are common in some localities, of 240 fecal samples of stray cats examined in kuwait, 22 (9.2%) were found to be infected with oocysts of coccidian protozoa (abdou et al. 2013) . toxoplasma gondii was found in 2.1%, and cats <6 months old had higher infection rate with oocyst of enteric protozoa than older cats. a serosurvey of the 240 stray cats revealed that 19.6% were positive to t. gondii igg. toxoplasma sero-positivity was observed in a higher number of adult cats compared to younger ones suggesting that with age the risk of exposure to t. gondii increases. thus, pregnant women handling cats and particularly kittens or cleaning out sand boxes have a chance of infecting their fetuses and eating raw meat. in pakistan, enteric pathogens are present not only in water but also foods contaminated from the environment or through human actions. mishandling of foods allows these pathogens to contaminate and multiply in them. for example, street-vended fruit salads, locally called fruit chats, offered for sale at high ambient temperatures without coverings, and khoya and burfi, two indigenous sweet dairy products, and locally produced ice cream are often heavily contaminated with enterobacter, e. coli, klebsiella, salmonella and s. aureus (akhtar 2015) . these contamination scenarios have led to outbreaks with cases severe enough to be hospitalized. bus and train stations where pulses (edible seeds of various crops as peas, beans, or lentils), ground meat dishes, and chickpeas are sold to passengers, and are also heavily contaminated with bacteria including clostridium perfringens. sweet dishes and home-prepared foods in small communities are commonly contaminated with s. aureus, c. perfringens, and bacillus cereus leading to rapid intoxications. one study confirmed campylobacters to be present in 48% of 1636 tested samples of milk and meats and 40.9% of vegetables in three major cities of pakistan (akhtar 2015) . a wide array of vegetables is routinely consumed in this country and serve as a rich source of vitamins, minerals, bioactive compounds, and fiber but these can be sources of enteric infections if they are consumed contaminated. shigella spp. has been shown to develop resistance and is generally thought to be a major cause of foodborne illnesses, especially among the poor where health care facilities are minimal; shigellosis is associated with poor sanitary conditions and unsafe water for drinking and preparing foods. possible etiologies can be postulated in the following outbreaks. unfortunately, it is not only pathogens that give rise to food-associated disease. soomro et al. (2008) highlighted the indiscreet use of pesticides in agriculture and its impact on environmental pollution. despite the increased production cost associated with extensive use of pesticides, their use is common in developing countries. numerous studies have demonstrated substantial levels of pesticide residues in various foodstuffs in pakistan, and the groundwater has been observed to be considerably polluted in many parts of punjab and sindh provinces of pakistan (akhtar 2015) . commonly used open rural wells in the punjab were polluted with six pesticides: bifenthrin, î»-cyhalothrin, carbofuran, endosulfan, methyl parathion, and monocrotophos. in the hyderabad region 61% of the tested samples of eight vegetables (cauliflower, green chili, eggplant, tomato, peas, bitter gourd, spinach, and apple gourd) were found to be contaminated with pesticide residues exceeding maximum recommended limits (mrls) (tariq et al. 2003; anwar et al. 2011) . heavy metals such as cadmium (cd), copper (cu), lead (pb), and zinc (zn) arising from increased industrialization can contaminate agricultural soils and these can be found in fruits (including widely-consumed mangoes), fruit juices, vegetables directly from soil uptake or from the processing and packaging (akhtar 2015) . for instance, spinach, coriander, and peppermint, grown in sindh province contained 0.90-1.20 mg/kg of arsenic resulting in a total ingestion of arsenic 9.7-12.2 î¼g/kg body weight/day in diet (arain et al. 2009; khan et al. 2010) . aluminum concentration in branded and nonbranded biscuit samples from hyderabad were found to range 7.4-84.3 and 34.5-70.2 mg/kg, respectively (jalbani et al. 2007) . similarly, javed et al. (2009) detected higher concentrations of cd, cr, ni, and pb residues (mg/l) in bovine and goat milk. pakistani foods are more prone to aflatoxin contamination because of the warm and humid climate, and the situation is exacerbated by malpractices during handling and storage of edible commodities (mobeen et al. 2011) . samples of broken rice, wheat, maize, barley, and sorghum ranged 15-45% with the highest aflatoxin concentration (15.5 î¼g/kg), in wheat samples (akhtar 2015) . chilies are widely eaten and exported, but aflatoxin levels can be eightfold higher than the eu permissible limits to pose a potential health risk to pakistani consumers; concentrations can be reduced by more appropriate care and handling of the chilies at pre-and postharvest stages. nuts and dried fruits in pakistan are cultivated and processed in the northern areas and have been shown to have aflatoxin levels above the eu limit of 4 î¼g/kg in up to 70% of samples (ahmad et al. 1989; luttfullah and hussain 2011) . aflatoxin m1 in milk and milk products requires regular monitoring in pakistan since 3% of the total tested samples of milk were found to exceed the us tolerance limit of 0.5 î¼g/l (hussain and anwar 2008; hussain et al. 2010) , and buffalo milk had higher levels of aflatoxin compared with cow's milk. intentional deception of consumers by blending low cost and inferior quality ingredients to make more profit of food intended for sale is prevalent in pakistan, where families are exposed toxic dyes, sawdust, soapstone, and harmful chemicals in beverages, oil or ghee, bakery products, spices, tea, sweets, bottled water, and especially milk and milk products where more than 50% of samples tested have had adulterants added (akhtar 2015) . one of the more innovative research projects to provide more home-grown food is in qatar. the sahara forest pilot (sfp) pilot study demonstrated that there are significant comparative advantages using saltwater for the integration of food production, revegetation and renewable processes: (1) seawater cooling system for greenhouses supports production of high-quality vegetables throughout the qatari summer, and reduces freshwater usage to less than half that of comparable greenhouses in the region; (2) solar and desalination technologies were successfully integrated as designed into the sfp system, such as the greenhouse and evaporative hedges providing wet-cooling efficiencies without cooling towers; (3) the external evaporative hedges provide cooling of up to 10 c for agricultural crops and desert revegetation with vegetable and grain crops growing outdoors throughout the year; (4) commercially interesting algae showed good tolerance to heat and high evaporation rates in the leftover salty water (miss22 2013; clery 2013). the concentrated solar power plant uses mirrors in the shape of a parabolic trough to heat a fluid flowing through a pipe at its focus. the heated fluid then boils water, and the steam drives a turbine to generate power. hence, the plant has electricity to run its control systems and pumps, and can use any excess to desalinate water for irrigating the plants. in summary, sfp allows food production in all 12 months of the year (3 crops) with half the fresh water usage than in comparable greenhouses. on the basis of the pilot success, sfp is now engaged in studies aimed at building a 20-hectare test facility near aqaba in jordan, large enough from the 1-hectare operation in qatar to demonstrate a commercial enterprise. tourism is popular in several middle eastern countries, particularly beach and coastal resorts in egypt and turkey. tourism has been the major economy in egypt for many years but can be threatened not only by civil unrest and terrorism but also by foodborne illness (costa 2008 ). tourists might not stop coming to egypt due to a few reports of diarrhea; however, widespread reporting of severe cases, and lawsuits, will make tour operators much more selective, and bring pressure on the egyptian hospitality industry to improve its hygienic standards. the greater challenge is for egypt to ensure that it has the capacity to sustain a safe food supply for its own people. in doing so, it provides safe food for those who want to explore its rich history and seaside resort areas. multiple reports of illness have been reported from nile river cruises and a resort town on the coast. from september to november, 2008, 34 cases of hepatitis a imported from egypt were reported to the german public health authorities (bernard and frank 2009) . investigations pointed to a continuing common source of infection, most likely linked to nile river cruises. in addition, eight cases from france had been travelling on a nile cruise and one on a red sea diving safari (couturier et al. 2009 ). one specific cruise ship was mentioned by six of ten belgian cases (robesyn et al. 2009 ). those who took a nile cruise had typically done this in combination with a hotel stay. at least three different ships and three different hotel accommodations were mentioned in the travel histories of the french cases. the patients affected had not been vaccinated, which emphasized the need for more effective travel advice before trips to hepatitis a endemic countries (sane et al. 2015) . possible sources of infection might have been contaminated food obtained from a common food catering company consumed onboard, contaminated tap water supplies for the ships' bunkers, or a common exposure on shore (e.g., a restaurant where tourist groups from various ships were taken during day trips). as all of these ships continuously traveled up and down a short stretch of the river (aswan to luxor and back) with standard mustsee stops along the way, the cases possibly shared an exposure on land. both the long incubation period of hepatitis a (15-50 days) and long delays in collecting information on the individual cases precluded any rapid intervention on location. no specific food source was identified but it could have been juices as recognized in an earlier major outbreak. in 2004, tourists returning from egypt included 351 hepatitis a case-patients from 9 european countries who were infected with a single hav strain (genotype 1b) (frank et al. 2007 ). the case-control study identified orange juice most likely contaminated during the manufacturing process, e.g., by an infected worker with inadequate hand hygiene or by contact of fruit or machinery with sewage-contaminated water. citrus fruit and citrus juices have occasionally been implicated as vehicles of hav and salmonella infections, with contamination typically occurring during production, or preparation just before consumption. as hav is resistant to acid, it likely can survive for prolonged periods in orange juice. it is also possible that leafy greens could contribute to foodborne illness in egypt. an international study of contamination of 562 leafy green lettuce and spinach samples taken between 2011 and 2013 from 23 open-field farms in belgium, brazil, egypt, norway, and spain showed that the egyptian samples were the most contaminated at 55.6% (liu et al. 2016) . these authors claimed that temperature had a stronger influence than did management practices on e. coli presence and concentration. region was a variable that masked many management variables, including rainwater, surface water, manure, inorganic fertilizer, and spray irrigation. temperature, irrigation water type, fertilizer type, and irrigation method should be systematically considered in future studies of fresh produce safety. also in the spring of 2008, a young couple was ill with vomiting and abdominal cramps after their first meal at a sharm el sheikh 4-star hotel in the egyptian coastal resort area, and they remained there in their bedrooms for the rest of their week (this is staffordshire 2008). both continued to have ongoing issues 6 months later, with one of them suffering from reactive arthritis. other guests also complained about diarrhea. they stated that the food was disgusting; the meat was undercooked, the buffet was left out for long periods of time, with new food being piled on top of the old food, and there were flies landing on food items. in august 2014, a family stayed at a resort hotel, also in sharm el sheikh, and all suffered severe symptoms including diarrhea, stomach cramps, and vomiting. they were put into the hotel clinic given antibiotics and intravenous drips but had not completely recovered after they returned home (galley 2014) . at the time other guests were also ill. they noticed that the food including chicken and beef, appeared to be undercooked a couple of times, and that one of the chefs touched raw meat and then touched cooked meat without changing gloves. the booking company confirmed that "a very small number" of guests staying at the resort in 2014 reported that they had been unwell, "with symptoms similar to a virus". the company said that guests were offered the appropriate support and advice by their overseas holiday advisors. it claimed that all of its hotels were subject to stringent monitoring and audits and this hotel achieved an extremely high score in its audit carried out in the summer of 2014. however, high audit scores do not necessarily correlate with day-to-day safe hygienic practices (powell et al. 2013) . the popular beach resort of sarigerme, turkey, on the aegean sea also has had a reputation for gastroenteritis, with repeat problems of foodborne illness with british tourists on vacations organized by tour companies, although the actual hotels were different. in 2009, an outbreak of gastric illness at this resort led to â£1.7 m paid out in compensation, with 595 people suffering from infections including salmonella, cryptosporidium, campylobacter and e. coli (hutchison 2015) . in september, 2012, hundreds of british holidaymakers suffered from salmonellosis after returning from a hotel complex in sarigerme (disley 2012) . final figures may have been close to 1000, and several were hospitalized. in october 2014, the swannell family had booked a week's stay at the first choice holiday village resort in sarigerme, when mark swannell, 46, fell seriously ill a few days into the break with diarrhea, abdominal pain, nausea and lethargy (hutchison 2015) . he said that some of the food he was served at the hotel had been undercooked, with some chicken bloody in the middle, food was not served at the correct temperature, food was left uncovered for prolonged periods of time, and the same food had been served more than once. the family stated that cutlery, crockery and table linen used in the restaurant was not up to standard, and they saw cats in the public areas of the hotel and in the restaurant. legal action was taken. in addition to ill tourists in middle eastern countries, contaminated exported food can affect those abroad, as illustrated in the following u.s. outbreak. from march to august 2013, of 165 patients identified with hepatitis a in ten states, 69 (42%) were admitted to hospital, two developed fulminant hepatitis, and one needed a liver transplant, but none died (collier et al. 2014) . almost all cases reported consuming pomegranate arils (seeds) from one retail chain. hepatitis a virus genotype ib, uncommon in the americas, was recovered from specimens from 117 people with hepatitis a virus illness. pomegranate frozen arils imported from turkey were identified as the vehicle early in the investigation by combining epidemiology, genetic analysis of patient samples, and product tracing. the product was then removed from store shelves, the public warned not to eat the seeds, recalls took place, and post-exposure prophylaxis with both hepatitis a virus vaccine and immunoglobulin was provided. this investigation showed that modern public health actions can help rapidly detect and control hepatitis a virus illness caused by imported food. egyptian trade has also been adversely affected by exports. in 2013, there were three outbreaks of hepatitis a sickening 400 persons in 16 -european countries. in the first report in april, 106 persons in four scandinavian countries were infected with hepatitis a (andrews 2014) . epidemiological investigations traced those cases to frozen strawberries grown in egypt and morocco, though no strawberries were found to be positive for hav. the second outbreak in april was larger in extent with 107 ill in 14 countries, all having recently visited egypt, and the outbreak strain of the virus had the same subgenotype as the first outbreak associated with strawberries. an epidemiological investigation into the second outbreak suggested the likely source was strawberries or another fruit distributed to hotels in egypt. the third outbreak was reported in germany in may, after nine germans were infected with hepatitis a after traveling to italy. this third outbreak infected about 200 italian residents, as well as nine germans, one dutch traveler and five polish travelers; 21 irish residents with no travel history to italy were infected by the same strain of the virus. separate investigations in italy and ireland both implicated imported frozen mixed berries as the source, with most of those berries coming from eastern europe. it is not known if these berries came from other regions, such as egypt, or were local to eastern europe. contributing factors to the larger number ill was lack of vaccination. because hav infections were declining in europe over the last few decades, fewer people had developed antibodies to repel the virus. couple that with the fact that hepatitis a was not on the vaccination schedule for citizens of many of the countries affected, and the result was a highly susceptible population. also, most of the european travelers to egypt were not advised to get hepatitis a vaccinations when staying in all-inclusive resorts, which were attracting an increasing number of europeans traveling to egypt. further, the investigators believe contamination of the berries occurred early in the food production chain. investigators suspect that irrigation water contaminated with sewage water likely contaminated the strawberries in the two outbreaks connected to egypt. but the contamination might have also been caused by infected workers in the field or the processing facility, or by contaminated water sprayed on the berries sometime before distribution. the outbreaks indicate that fresh and frozen berries are efficient vehicles of hav infection, as previously demonstrated in the us and elsewhere (palumbo et al. 2013) . european authorities agreed that "the 2013 experience demonstrated the absolute necessity for extensive collaboration between countries and between the public health and food sectors to identify as quickly as possible the vehicle of infection and, ideally, to control the outbreak in a timely fashion." a more serious outbreak damaged egypt's food export trade. in july 2011, the european union (eu) banned the import of certain egyptian seeds and beans till at least october following an official report that a single batch of egyptian fenugreek seeds probably caused two european outbreaks of e. coli infections responsible for 4211 ill persons and at least 50 deaths. a task force of health officials set up by the european food safety authority (efsa) reported that one lot of fenugreek seeds imported from egypt was the most likely common link between the two outbreaks in northern germany and in bordeaux, france (anderson 2011) . both were traced back a year and a half to a shipment of 33,000 pounds (15,000 kg) of fenugreek seeds, that was loaded onto a ship at the egyptian port of damietta on november 24, 2009. on the ship's arrival at antwerp, belgium, the seeds were barged to rotterdam to clear customs. the sealed container was trucked into germany to an unidentified importer, who resold most of the lot. an unidentified german company then resold about 150 pounds of the seeds to the german sprouter, which is believed to be the source of the sprouts that caused the extensive german outbreak. the german importer also sold about 800 pounds of sprout seed to the english company thompson & morgan, which repackaged the seeds into 1.75-ounce (50 grams) packages. those packages were shipped to a french distributor, who resold the seeds to about 200 garden centers around france. investigators believe that one of those packets was the source of the second european outbreak with 16 cases in the bordeaux area. because the seeds were likely contaminated with e. coli o104:h4 at some point before leaving the importer, and more contaminated seeds could be in circulation, it was deemed appropriate to consider all lots of fenugreek from the egyptian exporter as suspect. soil contact or animal or human fecal contamination of the seeds likely occurred during their production or distribution in egypt. even a negative laboratory test of those seeds could not be interpreted as proof that a batch was not contaminated. trace-forward findings indicate the german importer sold seeds from the suspected lot to 70 companies, and the shelf life of the seed can be up to 5 years. by mid-october, 2011, the european commission (ec) lifted import restrictions on fresh and chilled podded peas and green beans and other fresh produce from egypt, but the ban on egyptian seeds and sprouts, scheduled to expire on october 31, was to be extended until the end of march, 2012, following an "unsatisfactory audit" of seed producers in egypt (news desk 2011). the extended ban involved arugula sprouts, leguminous vegetable sprouts (fresh or chilled), soy bean sprouts, dried (shelled) leguminous vegetables, fenugreek seeds, soy beans and mustard seeds. the ec audit showed that measures taken by the egyptian authorities to address shortcomings in the production of seeds that may be sprouted for human consumption were not sufficient "to tackle the identified risks." those shortcomings were not seen in the growing and processing sites for fresh peas and beans, and therefore those vegetables were no longer considered a food safety risk. there is no need for actual illnesses to occur to affect trade. recalls, seizures, and bans can be employed by importing countries if standards are not met, and force exporting countries like egypt to take action. for instance, in 1999 the ec suspended the import of peanuts from egypt due to the presence of aflatoxin in concentrations in excess of maximum levels specified in eu regulations (technical cooperation department 2003) . egypt is a major peanut exporting country and the european markets then accounted for 68% of its peanut exports. this decision was repealed on 1 december 1999 and was replaced by another decision, which imposed a requirement for certification to accompany every consignment and required systematic analysis of consignments and documentation by the importing member state. under this system only 18 egyptian exporters were allowed to ship to the eu. in august 2003, the 1999 decision was replaced by another decision that required the competent authorities in eu member states to undertake random sampling and analysis of 20% only of peanut consignments from egypt for aflatoxin b1 and total aflatoxins. this improvement came as a result of the efforts that the egyptian government put in complying with the requirements of the eu. to this end, the egyptian ministries of agriculture and land reclamation (malr) and ministry of foreign trade (moft) issued ministerial decree no. 2/2000, which covered all stages of production, processing, sampling and exporting of peanuts. the main provisions of the decree were: exported peanuts must be produced, inspected and prepared according to set scientific procedures; and exporters who violate the rules would be suspended for 1 year; the decree also established the legal limit for aflatoxin in peanuts in both the domestic and eu export markets. in the egyptian domestic market, the legal limit was 5 mg/kg aflatoxin b1 and 10 mg/kg total aflatoxin content. for the eu market, the legal limits were 2 mg/kg aflatoxin b1 and 4 mg/kg total aflatoxin content. in addition, the decree specified the sampling procedures that must be followed for export certification. in september of 2001 the food and veterinary office sent a mission to egypt to assess egypt's compliance with its certification system requirements. a number of recommendations on steps egypt should take to improve the control system of foodstuffs intended for export to the eu were made. in response, the egyptian authorities declared that they were taking actions to address the mission's recommendation. but to achieve that there was a need to coordinate among a number of egyptian agencies involved in the production and export of peanuts and aflatoxin control: malr, the central administration for plant quarantine (capq), the agricultural research center (arc), the ministry of foreign trade (moft), and the customs service. also a laboratory capable of testing for mycotoxins was necessary. alongside this; egypt had technical assistance from international organizations in order to build human and physical capacities necessary for achieving compliance. the action by the eu forces egypt to improve the safety of its peanut production which would be beneficial both to europeans and to all who eat products made from egyptian peanuts, including the domestic consumers. lebanon used to be a tourist haven but is less today because of a seemingly dysfunctional government following a civil war. the country produces food for both the domestic and overseas markets. unfortunately, some exported food has caused illnesses and recalls. twenty-three cases of salmonella bovismorbificans in eight states and in the district of columbia (washington, d. c.) from august to november, were linked epidemiologically to hummus eaten at three mediterranean-style restaurants in the d. c. area, all owned by the same individual (goetz 2012) . although samples collected from all ingredients used to make the hummus tested negative for any salmonella, the hummus was recalled and the outbreak ceased. during its investigation of the restaurants, the d.c. department of health discovered multiple food safety violations at the establishments, including inadequate food temperature control, insufficient hand washing, and the presence of pests and insects, which had to be corrected. it is not clear if any abusive temperature conditions could have allowed growth of the salmonella in the hummus. the public was not notified because by the time the hummus had been withdrawn from the market, there were no further cases. however, the contaminated ingredient in the hummus was not discovered until may, 2012, when a traceback by the u.s. food and drug administration (fda) revealed that the tahini used to make the hummus in one of the restaurants had recently been associated with recalls in canada for contamination with s. cubana (september 2011) and s. senftenberg (february 2012). all tahini linked to these outbreaks had been imported from the same company in lebanon. the fda then mandated that all tahini products coming from this lebanese company be tested for salmonella before entering the u.s. and has recommended that u.s. and canadian officials partner to inspect the tahini manufacturing plant. this was the first time s. bovismorbificans had been implicated in a tahini outbreak in the u.s. as a result of this outbreak, the author stated it is important for public health officials and consumers to be informed that products made with imported sesame paste have been shown to be associated with salmonella outbreaks and that they should be considered as possible sources for foodborne illness in the future. in fact, contaminated sesame seed paste was in the news a few days before a cdc report on the outbreak was made public, after a supply of contaminated tahini was stolen from a california importer's warehouse, where it was being stored because a sample had tested positive for salmonella. the tahini, which had also been imported from lebanon but from a different manufacturer, was awaiting destruction, and the fda warned the public that the stolen, potentially contaminated tahini may be on the market. lebanese tahini has been implicated in several outbreaks in the past and subject to recalls (harris et al. 2015) . government oversight of the food industry is variable across the region with many regulations stemming back to colonial days, but modernization changes are gradually being considered or implemented. unfortunately, where some middle eastern countries are slowly moving forward to improve food safety, others are slipping back in their oversight because of conflict and lower public health priorities. there are relatively few large food processing operations except those managed by multinational companies, and most of the government oversight is on smes particularly small foodservice outlets. the states in the gulf cooperation council (gcc), each have an aggressive food safety policy but do not always follow identical approaches, some of which are well-established and some of which are innovative. the ksa has had a food inspection system in place for many years with reports of outbreaks published regularly, though no doubt it could be improved with more cooperation between the ministry of health, the municipalities and the saudi food and drug authority (sfda). the sfda was established under the council of ministers resolution no (1) dated january 1, 2003, as an independent body that directly reports to the prime minister (el sheikha 2015). the sfda is responsible to regulate, oversee, and control food, drug, medical devices, as well as set mandatory standard specifications thereof, whether they are imported or locally manufactured. the control and/or testing activities can be conducted at the sfda or any other agency's laboratories. moreover, the sfda is in charge of consumers' awareness on all matters related to food, drug and medical devices and associated other products and supplies. the sfda has to negotiate with the moh their mutual responsibilities following specific foodborne disease instances or consumer complaints. bahrain claims to have one of the more advanced food control systems in the region. in july 2008, as ambient temperatures heated up, the ministry of heath urged people to make sure the food they consume is properly stored during the summer months to avoid microbial growth and risk of food poisoning, e.g., keeping meat and fish at 4 c and to cook food thoroughly (haider 2008) . the ministry was aware that both visitors and locals want to eat safe food, especially as bahrain is moving towards more tourism with people are eating out more often. the ministry ordered shops to provide appropriate storage facilities, e.g., coolers and refrigerators, for food as part of its efforts to protect the public's health. inspectors were checking food stalls, ice-cream parlors and vegetable shops to ensure that customers were not being sold contaminated or rotten products. the ministry claimed to thoroughly investigate any complaints it receives, and to facilitate this a new hotline number was launched by the ministry for general public to report food contamination complaints against supermarkets, restaurants, coffee shops and hotels. specific advice for consumers included: being careful when buying salads; fruits and vegetables should be washed thoroughly before they are consumed; and dairy products such as milk, cheese and eggs, should always be refrigerated, since microorganisms grow faster in these products. the ministry claimed that bahrain has one of the best food control methods and food safety records in the region, and could even act in the future as a consultant in this field for other countries, including other gcc states. by 2015, government oversight had stepped up. in april, the ministry of health warned people against buying food advertised on social media or sold on the street by unlicensed retailers in bahrain, either made in people's homes or by street hawkers (anonymous 2015c) . the ministry stated that control of these home operations is difficult if someone suffers from food poisoning since inspectors are not allowed to go into homes. many homes sell food without a license and some would-be entrepreneurs even have barns where they slaughter livestock and market the meat illegally. there were 54,968 inspection visits conducted in 2014 by 25 inspectors from the food safety and licenses group, which closed 41 of around 7000 registered outlets. inspections cover imported food from ports right up to where it reaches restaurants and food outlets; 71,886 visits revealed around 883,584 tonnes of imported food were permitted for consumption, but 1873 tonnes were considered as non-consumable (rejected), during the same period. one of the more recent important programs is the smart inspection project launched in april 2012. inspectors, many with masters and phd degrees, visit restaurants and coffee shops to take food samples, as well as explain to staff how to store food and ensure its safety (anonymous 2015c) . it includes awarding food outlets that achieve a 100% food safety standard a blue sticker, while those meeting 80% of standards get a green sticker. outlets that fail to achieve basic standards are warned with a red sticker. the total number of outlets assessed between august 2013 and february 2015 was 241; 17 were presented with blue stickers, 174 with green stickers and 50 with red stickers. this project features daily inspections and is focused on small food outlets, some of which have caused food poisoning in the past. inspection visits depend on the hygiene of each outlet and the complaints received about them; some require two or more visits annually. high-level restaurants already have certified inspectors for evaluation and most of them require only one visit per year. the ministry's ultimate goal through this project is to decrease cases of foodborne disease, particularly important as bahrain is increasing its tourism efforts and, thus, ensuring food safety is essential. to support the ministry's initiatives, live demonstrations on food safety practices were promoted in kitchens in hypermarkets. however, if red sticker facilities fail to take advantage of educational material, they may be punished for neglecting food safety standards and guidelines though public prosecution. in a bid to improve standards of hygiene in restaurants, qatar's supreme council of health (sch) increased the number of spot checks on food outlets and has launched a hotline for residents to report food poisoning (walker 2014 ). the council is responsible for monitoring food establishments and implementing qatar's food laws along with the ministry of municipality and urban planning (mmup/baladiya). the sch embarked on an intensive inspection campaign, collecting food samples from all restaurants and food outlets in the country including suppliers. the inspection teams, which include specialized doctors from the sch's communicable diseases department and the environmental health inspection department, also medically check workers responsible for preparing food to ensure they are not carrying infections. those found to be handling food in an unhygienic way would be immediately dismissed. following a hotline complaint call, a report is filed, a team from the sch visits the affected people, then inspects the related food outlet and collects samples for laboratory examination. the latest crackdown was in response to the illness of a family of four which suffered food poisoning after eating chicken, rice and salad at a popular turkish restaurant which was closed down because a medical report prepared by the sch's environmental health section confirmed that the outlet served contaminated food and violated health regulations. tests conducted in the central food laboratory at sch found three types of bacteria causing diseases in food served by the restaurant. medical tests on the victims also showed that they were infected by the same bacteria, as well as one of the restaurant workers. another popular turkish restaurant was closed for 2 months after it was found that several customers were treated in the hospital for food poisoning symptoms including intense nausea, vomiting and diarrhea. as part of the sch's new campaign, experts would undertake community awareness drives, and organize seminars and training sessions about food contamination to improve understanding among owners and workers in food establishments. other closures occurred because of serving food with moldy ingredients, rotten vegetables in the kitchen, insects in pasta, and generally violating the provisions of the food law. the mmup increased the number of spot-checks and naming and shaming erring establishments on its website in arabic. the 2014 amendments to the food law gave greater powers to authorities to fine and close down venues that break the law including temporarily closing down establishments if it has violated food safety and hygiene regulations, and also has the power to recommend severe penalties. a follow up to one of these closed doha turkish restaurants was after a trial when five staff were each been handed fines, jail sentences and deportation orders after they were found guilty of causing food poisoning to approximately 20 customers ill with vomiting, nausea and diarrhea (santacruz 2015) . the restaurant was accused of serving spoiled and unsafe food on october, 2014. an affected pregnant woman gave birth to her baby 2 months prematurely. the manager of the restaurant was fined approximately $2500 and sentenced to spend 3 months in jail while three other staff members were each fined approximately $2000 and sentenced to 1 month in jail. during an inspection it was found that another staff member did not hold the necessary health certificate and was subsequently fined approximately $1800 and also sentenced to 1 month in jail. as well as the staff members being sentenced to jail and fined, the court of environmental misdemeanours also found that the restaurant itself was guilty of causing the food poisoning outbreak, and issued the restaurant with approximately $8100 in fines and ordered it closed for a further 3 months. in other parts of the world these penalties would seem unduly harsh, as it would be difficult for this restaurant ever to recover financially. coupled with education, there has been recent enforcement blitzes on food establishments such as hotels, restaurants and bakeries by oman municipalities, and a leading bakery in muscat was closed down because of rats in the premises in late december, 2015 (staff 2015 . this led food safety experts and the public to call for stricter rules and heftier fines to be imposed after surprise checks conducted by the muscat municipality, especially when it was disclosed that nearly half the restaurants in the bausher area were not following food safety standards. surprise inspections by the muscat municipality at 125 restaurants in bausher found that around 53 restaurants did not meet food safety standards and were violating rules formulated by the municipality. also, in the same time frame, ibri municipality officials were forced to shut down 42 commercial shops and they destroyed more than 3000 km of outdated food in 2015. according to the municipality's officials, 698 health violation letters were issued throughout the year, as well as 541 warnings were issued to different institutions operating in the wilayat of ibri. there are no easily-accessible reports on government oversight in pakistan and inspection actions are more likely to be released to the public through the press. in 2015, the islamabad capital territory (ict), administration conducted a drive against adulterated food items with unannounced inspections of food outlets in different markets and imposed fines amounting to rs110,000 (about us$1000) on owners for unhygienic conditions at their premises including restaurants, cafes, bakers, candy (sweet) stores, and a hotel was sealed (app 2015) . cleanliness conditions at the outlets' kitchens were found unsatisfactory and unhygienic while workers had not been vaccinated against viral diseases. some business owners were also paying less to their workers in contravention of the minimum wages act. business owners were directed to improve cleanliness conditions and ensure food safety standards failing which strict action would be taken against them. a cattle market was also ordered to "beef up" its security. punjab, pakistan's most populous province, has a population that is more than double that of california, and lahore, the provincial capital, has a vast array of food outlets. from the available press reports, the punjab food authority (pfa) has a mixed record of oversight of food operations. a pfa team visited the polo ground restaurant at the race ground park and found expired food, blocked sinks and unhygienic conditions in the kitchen and food storage area in contrast to the claimed high quality standards by the management of the supposedly high-class restaurant (raza 2015) . the team faced resistance from the management but it managed to enter the kitchen for inspection. pfa officials said the kitchen condition was similar to that of an ordinary road-side eatery, dispelling general perception that restaurants serving the elite follow higher standards of hygiene and food safety. however, the pfa in lahore had received a complaint that an assistant food safety officer had received rs50,000 (about us$475) bribe from the restaurant owner so he could keep his restaurant open (anonymous 2015d) . another restaurant on peco road sealed by the pfa for poor hygiene and unsanitary conditions of its workers in the second week of march, was opened for business the very next day. typically, according to the pfa's standard operating procedure (sop), a restaurant sealed for the first time may resume business after a week. at the end of the week, the proprietor has to submit an affidavit assuring the authority that all problems pointed out by the food safety officer had been taken care of prior to reopening it for business. the pfa director general (dg) had constituted a three-member committee to probe the complaint of bribery but it was later shelved. similar situations occurred when restaurants that had reopened before the stipulated period for closure had expired. in the first week of 2015, a restaurant was fined rs25,000 (about us$240) for unhygienic conditions and lack of soaps in the workers' washrooms, instead of following the pfa sops of sealing the premises. the sops regarding duration of closure and required permission from the pfa dg were stated to be flouted openly. however, a pfa spokesperson denied any wrongdoing, and the sop was being observed to the letter. she said a written permission from the dg used to be mandatory in order to de-seal restaurants, but now an operations deputy director can also issue permission for it. she also stated that the restaurant on peco road had not reopened on orders of the pfa; its owner had de-sealed it illegally. these reports indicate that there may be some illegal activities including bribery by inspectors but miscommunication on how much leeway inspection staff have on prevention and control practices may be more of the issue. in mid-2015 ayesha mumtaz became the new operations director of the pfa, tasked with ensuring food in punjab is unadulterated and safe (reeves 2015) . her self-declared war on unhygienic food generated so much publicity in the last 6 months that she became a household name in pakistan. mumtaz says many food producers know nothing about hygiene but are willing to learn. there's also a hardened mafia who are only interested in profit, she says. everyone in the street seems to know about mumtaz. storekeepers begin shooing away customers, hauling down the shutters, and heading into the shadows in the hope that mumtaz's scrutinizing eye will not fall on them. these traders would sooner lose business than risk a visit from a woman whose campaign to clean up the kitchens and food factories of pakistan has made her a national celebrity. she declared that the pfa cannot allow them to get away with their "perverse" activities and to "play havoc" with the lives of the people. consumers are unaware that the cakes and sweets that they buy over the counter are produced amid unhygienic conditions. she has found spoons encrusted with filth, fly-blown cans of gooey liquid lying around haphazardly, dirty containers, grimy rags and rusty tin cans, moldy scraps of cake, all involved in making cakes and sweets to be sold to the public. civil servants in pakistan are often accused of being lazy and corrupt. mumtaz is being feted as a rare example of a government official who actually champions the public's rights. she and her inspectors have so far raided more than 13,000 businesses, and pakistanis seem to approve. her fans call mumtaz the fearless one. hundreds of thousands have clicked like on the pfa's facebook page in appreciation of her work. there was a very famous hotel in the heart of lahore that she inspected and found the chiller where they keep all the foods together (vegetables with chicken, meat), but also a big rat; this became big news for the public. however, there are complaints that she does her raids with police and cameras to be broadcast nationally even before the owners are convicted, according to the lahore restaurant association. in 2011, the abu dhabi food control authority (adfca) planned to check all food handlers by 2012. the authority's emirate food safety training (efst) program, started in 2008, provides basic training in food hygiene and safety to those who work in food outlets (olarte 2011) . according to the adfca, small catering businesses in most countries have the lowest standards of food safety, and most workers in abu dhabi's 2500 small restaurants are illiterate and do not speak fluent arabic or english, making it a challenge for them to understand and follow safety guidelines and regulations; 69% of managers and 73% food handlers in the capital speak south asian languages such as urdu, hindi and malayalam (pennington 2014) . the training is now offered in four languages -english, arabic, urdu and malayalam -which the majority of food service personnel speak, and covers basic food hygiene issues including staff hygiene, food temperature, cross-contamination, cleaning and sterilization. to help them understand and follow food-safety rules, the adfca is using photographs to teach employees how to handle food safely according to international standards. the scheme is an extension of a pilot involving 600 small restaurants carried out in 2012-2014. as part of the efforts to ensure retention of their learning, the adfca conducted spot checks at 94 food outlets in marina and khalidiya malls, and gave guidance and advice to staff for those with violations, rather than just penalizing them, the normal practice in most middle eastern countries. the field operations manager at the adfca noted that the differing cultures, education and languages are the barriers that sometimes hinder food handlers from carrying out what they are trained to do. he recommends that supervisors should quiz them on hygienic and safety issues so that they know how to properly prepare and serve food. those who have learning difficulty or are illiterate are given assistance through illustrations, in order to make it through the lessons and pass the examination. one of the critical elements of food safety that the adfca has to monitor and ensure, is that food handlers are aware of cold ready-to-eat food being kept at 4 c, while hot food should be kept and served very hot > 60 c. the adfca categorizes the food premises and carries out inspections based on their risk factors -high, medium and low. restaurants and hypermarkets belong to the high-risk group; warehouses to the medium risk; while groceries, honey shops and vegetable and fruit outlets are considered low risk. recently, the establishment of the egyptian food safety authority was initiated by the minister of trade and industry, with the support of the ministry of health and the ministry of agriculture. it would be responsible for food safety and consumer protection through the provision of sound data and guidance to deal with processed or genetically modified food in accordance with food safety standards (anonymous 2012b) . the strategic plan for the new draft law includes a revision of all egyptian laws and legislation that deal with food safety since 1893, including around 2446 other legislations. the authority would need to apply food safety standards on imported food the same way it does for locally produced foodstuffs. adopting the draft law would in effect cancel all existing laws and create one food safety law for the country. the food safety authority plans to monitor the foods consumed by egyptians of different age groups as a basis for where to put resources. another issue to be faced is that studies in egypt based on us statistics have revealed that the cost of food spoilage costs the country 144 million egyptian pounds annually. the chamber of food industries indicated that a unified body for food safety to apply international quality specifications and unite regulators was lacking. this reduced the competitiveness of local products, especially since most foreign countries do not recognize egyptian regulations. it was hoped that investors in food industries would bring in new investments to the sector in the upcoming period if a food safety authority were to be established, as per a ministerial decision issued in 2011. the food safety authority has received several approvals from governments that ruled during the 4-year period following the revolution, but apparently nothing has been yet finalized until recently (mefreh and saeed 2015) . in a similar way to egypt, the lebanese government has been debating a new law on food safety for many years but unlike egypt, it has yet to make much progress. lack of agreement at the parliamentary level has resulted in different ministries (health, agriculture, industry, environment, tourism) taking action as they see fit. the latest was in november 2014, when the minister of health conducted an extensive campaign of inspections in lebanese establishments and naming of facilities that did not meet the ministry's expectations (naylor 2014) . the minister personally revealed that numerous supermarkets, bakeries, butchers and restaurants had been violating food safety and sanitation standards. they shut down slaughterhouses, restaurants, supermarkets and other retailers selling contaminated food. for instance, changes needed to be made for the slaughterhouse to conform to health standards; the report said livestock must be hanged during slaughter and not laid on the ground and that the abattoir should also be equipped with refrigerators and storage units for separate types of meat and their cuts. however, discord among ministries is apparent with the tourism minister trying play down the publicity of the health minister's food safety blitzes by saying "we are in favor of full transparency, but we feel like we were 'deceived' because the food safety situation in lebanon is good and better than other countries. we apologize to tourists, but more importantly, any of the ministry of health staff is ready to apologize to the lebanese citizens for the public sector's failures throughout the years?" (yaliban 2014) . foodborne disease surveillance is limited in lebanon and cannot be used to indicate the actual level of foodborne illnesses in the country. lebanese food exports are also being required to conform to international standards. tahini made from sesame seed paste is a major food export to the west, but recalls of tahini manufactured in lebanon because of salmonella contamination are more frequent than they should be; one recent example was a health hazard alert for certain clic, al nakhil and al koura brand tahina products that may have contained salmonella, recall/advisory dated august 16, 2013 posted from canadian food inspection agency [also see tahini/hummus linked illnesses under foodborne disease in specific countries]. under the new us food and drug administration food safety modernization act, foreign companies importing foods to the us must demonstrate that they have the operational plans and facilities sufficient to produce safe food before they can ship any product to the us (fda 2015), which is causing some concern among lebanese tahini manufacturers and government agencies. thus, although there is knowledge about foodborne disease and other food safety issues within government, industry and academia, the political inertia means that many foodborne illnesses will continue to occur but not be properly reported or know what factors were present to cause the outbreaks. industry currently is taking the lead; apart from companies promoting food safety like boecker and gwr food safety, mena food safety associates (mefosa) (http://www.mefosa.com/), based in beirut, assists mena companies hone their competitive edge by establishing and verifying procedures and practices that ensure quality, wholesome and safe products through consulting, auditing and training services in haccp, gmps, and hygienic practices. however, lebanon's lack of a coordinated system of government oversight of the food industry pales into insignificance compared to that in syria. prior to the war, syria's healthcare system had hospital and doctor levels equivalent to other middle-income countries such as brazil, turkey and china, with life expectancy of 76 years, and most of the disease burden being similar to that in the west with non-communicable diseases, but four years of violence have changed all of that. child vaccination levels dropped from 90% pre-conflict to 50% in march 2014 (templeton 2015) . as a result, outbreaks of diseases that had long been under control have spread across the land and into neighboring countries: hepatitis, measles, leishmaniasis, multi-drug-resistant tuberculosis, typhoid and even polio, which had not been seen in the middle east for 20 years. life expectancy has dropped by two decades. medical personnel are clearly targeted because they are seen as potential enemies helping the opposite side. the majority of syria's doctors have been killed or fled the country (>700 medical workers have been killed since 2011). the situation has been called the worst humanitarian catastrophe this century, and the worst concerted attack on healthcare in living memory. at least 300,000 syrians have been killed and more than 11 million others have been forced from their homes since the conflict began on march 15, 2011, with over four million people in areas that are hard to reach for humanitarian aid, and 4ã�5 million have fled mostly to neighboring turkey, lebanon, jordan, and northern iraq, while others have sought safety in europe, provoking a political crisis in the 28-member bloc (devi 2016) . another middle eastern country under stress but with less publicity is yemen. currently there is little government oversight into food as there is little to be had. the situation in yemen is characterized by large-scale displacement, civil conflict, food insecurity, high food prices, endemic poverty, diminishing resources, and movement of refugees and migrants (wfp 2016). the un world food programme (wfp) has been in yemen since 1967. in 2014, wfp conducted a comprehensive food security survey which found that 41% of the people (10.6 million) were food insecure, of which some five million were severely food insecure, meaning they were unable to buy or produce the food they need to survive. the organization's protracted relief and recovery operation (prro), aims to reach six million people between mid-2014 and mid-2016 with 366,734 metric tons of food and us$74.5 million in cash and vouchers at an overall cost of us$491 million. if the conflict continues, this goal is unlikely to be met in time since both the airport and shipping port are areas being fought over. the wfp has been attempting to bring in relief supplies but cannot do so under fire, which means that only small amounts are occasionally delivered to the country (mukhashaf and miles 2015) . one example of this occurred in aden on july 20, 2015 when a ship docked after waiting a month to unload enough u.n. food aid to feed 180,000 people for a month. previous repeated attempts to send ships to aden were been blocked due to severe fighting in the port area. the prro is aligning wfp's activities with moves to increase the government's capacity to respond to the crisis and will promote recovery and resilience to enable food insecure households and communities to better withstand and recover from the effects of conflict and shocks. there are many similarities as well as substantial differences in the descriptions of issues concerning food safety and foodborne disease of each country in the region. gastrointestinal diseases are frequent throughout the middle east with some countries identifying their etiologies, such as egypt, kuwait, israel, pakistan, turkey, yemen. these include bacteria and parasites, e.g., salmonella, shigella, campylobacter, enterotoxigenic e. coli (etec), giardia, entamoeba, and occasionally enteric viruses such as hav and norovirus. however, none of the countries has a well-functioning foodborne disease surveillance system, but a few report on a regular basis like ksa, and starting recently, lebanon with pulsenet. mostly it seems that only large outbreaks or ones with fatalities that are reported on, and mainly through the press. these outbreaks are often related to point sources which are in most cases communal foods prepared for a large number of individuals as in feasts, student hostels, schools, campuses, or military camps. however, the actual etiological agents and the factors contributing to outbreaks are only rarely determined. one example is a very large outbreak in bahrain in 2002 with at least 750 people suffering from foodborne illness after eating contaminated egg-andmayonnaise sandwiches served at a wedding party, but the etiology was not determined, even though clinical specimens and food samples were analyzed, at least in a publically-released report (promed-mena 2002) . based on the type of preparation including the length of time taken for preparation of the implicated food and the time from consumption to the appearance of symptoms of foodborne illness, the types of symptoms, and what has already occurred historically in foodborne disease outbreaks, possible agents can be surmised, such as bacillus cereus and staphylococcal enterotoxins, and salmonella, shigella, or norovirus infections, but promed is continually asking for more information once an outbreak is announced, and hardly ever receiving it (promed-mena 2014). all this indicates that even if clinical specimens or food samples are taken and analyzed, laboratories are only rarely able to determine an etiologic agent, or at least report on their results. most agents described with the little information available seem similar in all the mena countries and to those encountered in the west. however, a few pathogens are more likely to be restricted to a few nations, such polio in pakistan, cholera in iraq, mers-cov in ksa, and botulism in egypt and iran where river fish are often eaten (one case of infant botulism was diagnosed in israel but it is a rare disease anywhere); the first two are more likely transmitted though water or poor hygienic conditions, the third by camels, and only botulism exclusively through food. brucellosis is widespread in the middle east but only a few country studies indicate its link to meat or dairy products. much of the middle east is in the throes of conflict which results in unique situations in specific countries to exacerbate foodborne disease or food poisonings; these include relief agencies supplying "stale" food to those trapped and starving by the syrian civil war, almost lack of food at all in yemen, deliberate poisonings of enemies in afghanistan, syria and iraq, accidental pesticide poisonings in iran, preventing unsafe food being sold to those on the hajj in ksa, improperly prepared catered food for foreign troops in bases in afghanistan, iraq, kuwait, ksa, and turkey. countries where tourism is a major source of income can be adversely affected by bad publicity over complaints over food served in resorts, such as in egypt and turkey. also, gulf countries tend to employ workers from india and other surrounding territories, and these are typically housed in camps or separate communities from citizens and visitors, and are transported to work sites and back; conditions are not always conducive to safe food, and outbreaks are occasionally reported either from their work sites or their overnight residences where meals are prepared or catered. most food to many of these countries is imported, especially those with limited agricultural land and adequate water supplies; fruits and fresh vegetables, tend to be grown in rural or peri-urban settings for local consumption and these can be contaminated at source through polluted river or well water, such as in the bekaa valley of lebanon and mountain communities in pakistan, and the nile, tigris and euphrates fluvial plains. on one occasion, iranian watermelons were recalled and future sales banned in ksa, qatar, and uae because they were suspected of being poisoned or were injected with pesticides (nobody claimed to be ill after eating the melons), because holes were found in a few of them. however, the rationale of iranian farmers deliberately losing money seems to counter this argument, and it is more likely a sectarian economic barrier (abdullah 2015) . in fact, with the temporary ban the price of watermelons went up in the countries that had banned them. random tests carried out on the fruit confirmed they were free from any chemical substances, insecticides or other pollutants. the holes were most likely caused by emerging insect pupae. countries outside the gulf region reported no problems with the imported iranian melons. where some processed foods are exported, there is a risk of the importing countries recalling these if they cause foodborne illnesses or contaminants are found in them. this has happened in egypt with hepatitis a virus in strawberries and e. coli o104:h4 in fenugreek seeds causing serious illnesses in europe and restricting further trade for an extended period. the same issue affected turkish pomegranate arils and lebanese tahini (made from imported ground sesame seeds), both containing salmonella, exported to the us. large to medium operations for broiler chickens and egg layers in ksa, kuwait, lebanon and other countries try and meet national standards or international guidelines for salmonella but are not always achieved, resulting in recalls and fines. governments are also aware of increasing concern over campylobacter in chickens, as widely-eaten poultry is a major source of this pathogen, but campylobacteriosis is not often cited as causing foodborne disease. raw milk (cow, sheep and camel) and raw milk cheese are still widely consumed in the middle east at the local level, though not usually obtained through supermarkets, and the risk of infections is high, as it is in other parts of the world, but with the added concern of brucella spp. and mers cov (the latter in the gulf countries where camels are bred and milked), both serious pathogens. yoghurt, surprisingly since it is acidic and is a source of gut beneficial lactobacilli, apparently was the foodborne vehicle to cause illnesses and deaths in afghanistan, israel, and pakistan. no agent was found in any of the samples. in the afghani example, the yoghurt was claimed to be deliberately poisoned; in the israeli one, it was apparently "stale" given to palestinian prisoners; there were two episodes in pakistan, one was from a home-prepared meal and the other from a restaurant which served rice and yoghurt. for prevention and controls strategies, most countries seem to rely on local authorities (municipalities) to do inspection of food facilities, more typically restaurants than processing plants as there are far more of them. illegal sales for unapproved products by local entrepreneurs are sometimes an issue, e.g., homeslaughtered meat in bahrain, and palestinians shipping food to israel. these illegal operations probably occur more often in porous borders within the region, and are only recognized when authorities decide to become vigilant in this area. some countries have conducted research and surveys much more than others based on the publication record, e.g., egypt, israel, palestine, ksa, turkey, and to a lesser extent, iran, lebanon, pakistan, uae, and yemen, but some research may occur without formal publication in recognized journals, making it difficult to have a true picture of how food safety problems are recognized and controlled. a few surveys have shown that home makers and food employees have limited knowledge of food safety, as in other regions. thus, some agencies or industry associations, sometimes in collaboration with outside organizations like fao or who, have attempted to train food employees in basic haccp principles, including best hand hygiene practices, and speakers give the latest food safety issues at the annual dubai international food safety conference, now in its 10th year. a few governments have established food safety agencies that have broad powers to inspect and control without overlapping responsibilities; these include jordan and ksa with food and drug administrations, uae with abu dhabi and dubai food control authorities, oman with its national food quality and safety centre, and pakistan with a punjab food authority. egypt and lebanon are initiating food safety authorities. israel, palestine and jordan have a cross-border agreement to collaborate on food safety issues. typical of many food control agencies in developing countries, periodic campaigns are launched to "crack down" on foodservice operations and sometimes processing plants. these are usually stimulated by complaints of the public, or the need for the responsible ministry to be seen doing something to justify its existence in compliance with regulations (if they exist). this has occurred recently in lebanon, qatar and pakistan. one issue is that poorly constructed or out-of-date regulations may be interpreted in different ways by the owners and the agencies (kullab 2014) . if a violation is found, the facility may be fined and/or temporarily closed down until it has satisfied the inspectors at the next visit. in one extreme instance in qatar, the owners and employees, were fined, imprisoned and deported. unfortunately, although the names of those at fault are often publicized by the media, their specific violations and how they relate to the regulations are not usually documented or at least publically released. another issue is that whether illnesses are suspected or not following a complaint, inspectors often insist that all food be discarded as soon as a sufficient violation, which may be unrelated to the complaint, has been determined; this prevents any samples being taken for outbreak investigations (hanna et al. 2009 ), as well as using the outbreak for a teaching tool for the owner and other similar operations. in conclusion, some progress has been made in the surveillance of foodborne disease in the middle east, but the disease's health and economic burden is barely being considered in many countries for future decision-making policies, an issue that is being tackled at the global level (who 2015b). food control agencies seem to be trying to stop apparent abuses but have limited resources to do much more. this region, in particular, is severely strained because of sectarian distrust, on-going civil wars, and terrorist attacks, with refugees from iraq seeking shelter toward europe but stalled in turkey and lebanon for long periods of time. the crisis in syria is considered the greatest humanitarian disaster of the twenty first century, or even since world war ii, and it looks like the on-going fighting including outside armed forces will make food insecurity in the affected countries even worse in the foreseeable future. less public attention has been directed to yemen where food insecurity is a major concern. this coupled with gulf countries losing their wealth over low oil prices and a resultant stagnant global economy means a focus on food safety will likely become lower in priority for many of these countries. since secure food has to be safe, as illustrated by "stale" food being issued to besieged syrian residents and prisoners, it is important that relief agencies and countries themselves be aware of the risk of foodborne diseases associated with immunocompromised persons, particularly children. however, even in countries where the food supply is acceptable, inadequate hygienic practices put the local and tourist population at risk of illness and exported foods jeopardize industry profits and a poor reputation for future trade. as demonstrated by ksa, jordan and uae, single agencies or multiple agencies with clear-cut roles responsible for food safety, should be pursued by governments in consultation with industry and academia. duplication creates ambiguities for enforcement and education strategies as well as being unnecessarily costly. water supplies are also critical and some governments are weaning away farmers from depleted groundwater aquifers, and making irrigation more efficient where there are sustainable supplies. water for irrigation and processing has to be both free of pathogens and unacceptable levels of chemicals, and effectively treated waste water can substitute for groundwater. the sahara forest project in qatar is one example of a very dry country using seawater resources effectively; an even larger project is being considered from the 1-hectare in qatar to a 20-hectare test facility in jordan (clery 2013) . all these issues are being compounded by climate change and expected higher temperatures in already arid lands, which will make the region all the more dependent on more expensive imported foods. gulf counties have enough petro-dollars to afford 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